Anti-Inflammatory Activities of Leaf Oil from Cinnamomum subavenium In Vitro and In Vivo

The study determined the chemical constituents and anti-inflammatory effects of leaf oil from Cinnamomum subavenium (CS-LO) that has been used in folk medicine to treat various symptoms including inflammation. The anti-inflammatory effects of the oil were evaluated by LPS-stimulated RAW264.7 cells and the Carr-induced hind mouse paw edema model, respectively. In vitro, nitric oxide (NO), prostaglandin E2 (PGE2), TNF-α, IL-6, and IL-1β were significantly decreased by CS-LO, and the expression of nuclear factor-κB (NF-κB) protein was blocked as well. In in vivo, the malondialdehyde (MDA) and paw edema levels were decreased by CS-LO, and the same result came up on the NO and tumor necrosis factor (TNF-a) of serum at the 5th h after Carr injection. In addition, iNOS and COX-2 immunoreactive cells of the paw tissue were decreased significantly by CS-LO (200 mg/kg) in histological examination. The present findings indicated that CS-LO have anti-inflammatory properties, and the effects might be caused through inhibiting iNOS, COX-2, TNF-α, IL-1β, and IL-6 expression via affecting NF-κB pathway, which will provide a power scientific basis for CS-LO to be used as the treatment of inflammatory diseases.


Introduction
The inflammation, associated with pain and swelling, is a series of the multiple biological reactions of body tissues to harmful stimulation. It is self-protective function of organisms to harmful stimuli [1]. Inflammation is used to be a hazardous factor for the onset of some chronic diseases, such as neurological disease, cardiovascular disease, and cancer [2]. Therefore, many studies considered anti-inflammation and antioxidant as a practical way to fight these degenerative diseases [3]. Plant extracts and essential oils, which are rich sources of antioxidant and anti-inflammatory agents, can suppress the release of inflammatory mediators and free radicals and increase antioxidant defenses. They are widely used in food, cosmetics, and medicine for their potential biological activities [4,5]. Therefore, the use of natural additives is a good way to fight abnormal inflammation and oxidation.
The anti-inflammatory effect of some Cinnamomum oil has been confirmed, such as Cinnamomum osmophloeum, Cinnamomum insularimontanum Hayata, and Cinnamomum cassia. Much of the ingredients of the essential oils belong to terpenes, including monoterpene, sesquiterpene, and their oxidative derivatives. These small molecules tend to diffuse through the membranes to induce biological reactions.

BioMed Research International
Moreover, the leaf oil of C. subavenium has potent antioxidant and antimicrobial activities [13]. But, the anti-inflammatory activity of C. subavenium has not been report so far. Therefore, the objectives of our study were to investigate the major chemical composition of leaf oil from C. subavenium using gas chromatography-mass spectroscopy (GC-MS) and assess the anti-inflammatory activities of the leaf oil from C. subavenium (CS-LO). . . Preparation of CS-LO. 500 g fresh leaf of C. subavenium was hydrodistilled for 4 h with a Clevenger-type apparatus. The collected oil, dried with anhydrous sodium sulfate, was stored in brown vials at +4 ∘ C.

Materials and Methods
. . Gas Chromatography and Mass Spectrometry Analysis. The constituents of essential oil were identified by gas chromatography-mass spectrometry (GC-MS). MS is equipped with Polaris Q quality selective detector in electron collision ionization mode (70 eV). The chromatographic column was an RTx-5 (25 m x 0.25 mm x 0.25 m), kept 1 minute at 80 ∘ C to 200 ∘ C at 4 ∘ C/min, and kept 5 minutes. The temperature of injector was 250 ∘ C, the helium gas flow rate was 10 mL/min, and the split ration was 1:10. Manual injection of diluted samples (1.0 l, 1/100, v/v, ethyl acetate) was in nonshunt mode. The major chemicals of CS-LO were identified by comparison with standard products and retention indices based on their mass spectral fragmentation in The Wiley GC-MS library. The amount of compounds was determined by Peak area of integral spectrograms.
. . Cell Viability Assay. The RAW 264.7 cells were obtained from Cell Culture Center of Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in DMEM supplemented with FBS (10%), streptomycin (100 mg/Ml), and penicillin (100 U/mL) at 37 ∘ C in a humidified atmosphere with 5% CO 2 and were subcultured every 3 days. The CS-LO was dissolved in DMSO and diluted with culture medium to target concentration. RAW 264.7 cells (1.5×10 5 ) in 96-well plates were treated with 0, 2.5, 5, 10, 20, and 40 g/mL of CS-LO in LPS at 37 ∘ C for 24 h. Adding MTT solution to every well, the cells were incubated for another 4 h at 37 ∘ C. And then, the medium was discarded; the formazan crystals were dissolved in DMSO. The absorbance was measured at 570 nm.
. . Detection of Intracellular NO, PGE , TNF-, IL-, and IL-. NO production was measured by the nitrite levels of the cultured media and serum based on the Griess reaction [14]. RAW 264.7 cells were incubated in 0.2 g/mL LPS with different CS-LO (2.5, 5, and 10 g/mL) for 18 h. Then, the collected medium was mixed with the same volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid). 15 min incubation later, the absorbance was detected at 540 nm using Micro-Reader. RAW 264.7 cells treated with LPS and CS-LO were incubated with DAF-FM (10 M) diacetate at 37 ∘ C for 1 h, and the production of NO was assessed by confocal laser scanning microscopy. The production of PGE 2 was quantified with the Prostaglandin E2 Express EIA Monoclonal Kit. IL-1 , IL-6, and TNF-were quantified with their ELISA kits under the manufacturer's instructions.
. . Western Blot Analysis. After washing with cold PBS, the stimulated RAW 264.7 cells were lysed in a cold lysis buffer [10% glycerol, 1% Triton X-100, 1 mM Na 3 VO 4 , 1 mM EGTA, 10 mM NaF, 1 mM Na 4 P 2 O 7 , 20 mM Tris buffer (pH 7.9), 100 mM b-glycerophosphate, 137 mM NaCl, 5 mM EDTA, and one protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA)] and incubated on ice for 0.5h. The collected supernatants were rapidly frozen. The concentration of the proteins was detected using the BCA method. After denaturation, the proteins of the cell extracts were separated by SDS-polyacrylamide gel electrophoresis. After electroblotted onto a PVDF membrane, they were incubated with blocking solution (5% skim milk) for 12 h at 4 ∘ C, and then incubated with primary antibody for 4 h. After washed with Tween 20/Tris-buffered saline (TBST), the blots were incubated with a dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were washed again with TBST and then developed using enhanced chemiluminescence. The quantities of western blot were made by measuring the relative intensity contrasted with the control using Kodak Molecular Imaging Software.
. . Carr-Induced Paw Edema. Male Wistar rats (180-220 g), two months old, were provided by the Hubei Provincial Center for Disease Control and Prevention. All animals were kept under 23-25 ∘ C conditions and 12 hours of light/12 hours dark cycles. The experiments and animal maintenance followed the animal care and use guidelines of the Institute's Animal Care and Use Committee of Huazhong University of Science and Technology. The Wistar rats, injected with 1% Carr (50 L) in the plantar side of right hind paws by 30min in advance, were administered with CS-LO (50, 100, and 200 mg/kg, p.o.), Indo (10 mg/kg, p.o.), and the vehicle (pure water + CMC 0.5%). The volume of paw was measured using a plethysmometer (model YLS-7B, Zhong Hao, Peking, China) before Carr injection and every hour after administration of the edematogenic agent for 5 h. The degree of edema (%) was measured by the ratio ((V1−V0)/V0), where V0 is the normal volume before injection, and V1 is the pathological volume after Carr injection. Finally, the rats were sacrificed; the collected blood and Carr-induced edema feet were stored at -80 ∘ C until analysis.
After being homogenized in PBS (pH 7.2) and centrifuged at 12000 × g for 5 min, the collected supernatants were stored at -20 ∘ C for MDA determination. The collected serum was stored at -20 ∘ C for the NO, PEG2, and TNF-assay.
. . MDA Assay. MDA of the paw edema tissue was measured with the thiobarbituric acid reacting substance (TBARS) [15]. In brief, MDA was used to react with thiobarbituric acid to form a red-complex TBARS under high temperature and acidic conditions, and then it was determined at 532 nm.
. . Histological Examination. Histological examination was performed based on the methods by Huang et al. [16]. Briefly, after being dehydrated by ethanol, the tissue sections were paraffinized. After deparaffinization, some sections (thickness, 7 m) were stained with hematoxylin and eosin (H&E); the others were processed for iNOS and COX-2 immunohistochemistry staining. BH2 Olympus microscopy was used to observe and photograph the samples. 3-5 tissue slices were randomly chosen from Control, Carr, Indo, and CS-LO-treated groups.
. . Statistics: Experimental. Data were presented as the mean ± standard deviation (SD) of three parallel measurements. The statistical significance was evaluated by oneway analysis of variance (ANOVA). Statistical significance is expressed as * p < 0.05, * * p < 0.01, and * * * p < 0.001.

. . Effects of CS-LO on RAW
. Cell Viability. The toxicity of CS-LO was determined in RAW 264.7 cells by MTT assay; the result is shown in Figure 1(a). Cell viability did not change at 0, 2.5, 5, 10, 20, and 40 g/mL of CS-LO. These results suggest that there were no toxic to RAW 264.7 cells under 40 g/mL of CS-LO. Therefore, CS-LO (2.5-10 g/mL) was used in the subsequent experiments.
. . Effects of CS-LO on LPS-Induced NO Production. NO production may reflect inflammation; the effect of CS-LO on LPS-induced NO in RAW 264.7 cells was assessed. After treatment with CS-LO and L-NAME (NO inhibitor), the level of NO in RAW 264.7 was evaluated. As shown in Figures 2(a) and 2(b), there was a concentration-dependent inhibition in NO production. The group treated with 10 g/L CS-LO (p < 0.01) was highly decreased compared to the LPS-alone group, and L-NAME (100 M) also have a significant inhibition on the NO in RAW 264.7 cells (Figure 2(a)). Moreover, the image of confocal laser scanning microscopy indicates that the NO production was also suppressed by CS-LO (Figure 2(b)).  . . Effects of CS-LO on the MDA Level. MDA was measured to evaluate the ability of CS-LO to radicals. As shown in Figure 4(b), there was a higher level of MDA in paw tissue at 5 h after Carr injection (p < 0.01). However, dose-related inhibition of the MDA level was observed in paw tissue at 5 h after treatment with CS-LO (50, 100, and 200 mg/kg); CS-LO at 100 and 200 mg/kg significantly decreased the MDA level in the paw tissue (p < 0.01), the same with Indo (10 mg/kg) (Figure 4(b)). production, (c) TNF-production, (d) IL-1 production, and (e) IL-6 production. The data represent the means ± SD values from three independent experiments. * P < 0.05, * * P < 0.01, and * * * and P < 0.001 compared to the LPS-treated cells alone. level increased significantly in the serum of rats. However, the inhibition of CS-LO to NO in the serum of rats was found with a dose-dependent manner, and 200 mg/kg of CS-LO significantly reduced the NO level in the serum (p < 0.01). Figure 4(d), the TNF-level in the serum increased significantly at 5 h after treatment with Carr (p < 0.01). However, CS-LO highly reduced the TNF-in the serum, and 200 mg/kg of CS-LO significantly reduced TNF-level in the serum (p < 0.01). Figure 4(e), the PGE 2 level in the serum increased significantly at 5 h after treatment with Carr (p < 0.01). However, CS-LO decreased PGE 2 in the serum with a dose-dependent manner, and CS-LO (100 and 200 mg/kg) highly inhibited the level of PGE 2 (p < 0.01). . . Histological Examination. The histological appearance showed a normal appearance of tissue without any significant lesions in the control group ( Figure 5(a)). Haemorrhage and a large number of neutrophils infiltrated the interstitial tissue in Carr-treated group ( Figure 5

Discussion
Inflammation is mediated by a series of free radicals and inflammatory cytokines. Excessive or persistent generation of these inflammatory mediators may cause diseases, such as arthritis, diabetes, atherosclerosis, and some types of cancer [17]. During inflammation, the activated macrophages are the source of inflammatory cytokines, which were important cells for phagocytosis and molecular immunology [18]. Moreover, many researches showed that the inflammatory free radicals could be induced by l-carrageenan (Carr), which was the suitable way to study the inflammatory effect. Previous studies had demonstrated the bioactivity of the C. subavenium. stems of C. subavenium had good pigmentation inhibitory abilities [12]. Cheng et al. found that the leaves and stems of C. subavenium had antioxidative activities [19]. Ho et al. reported that the leaf oils of C. subavenium had good antimicrobial activities [13]. Chen et al. reported the extraction of C. subavenium leaves had inhibition to various cancer cell lines [20]. So in this study, we analyzed the leaf oil of C. subavenium activity may be due to the effects of minor ingredients or synergetic effects among the ingredients. The proinflammatory cytokines NO and PGE 2 plays important roles in inflammatory diseases. NO is an endogenous free radical substance synthesized from L -arginine by nitric oxide synthase (NOS) in various animal cells and tissues [21]. PGE 2 is mediated by cyclooxygenase (COX), which has COX-1 and COX-2 isoforms. Therefore, the inhibition of NO and PGE 2 is an effective strategy for inhibiting inflammation [22]. Our result showed that the production of NO and PGE 2 was significantly inhibited by CS-LO ( Figures  2 and 1(b)); we also found that CS-LO downregulated iNOS and COX-2 with immunohistochemical staining (Figures 5(h) and 5(l)). Thus, the anti-inflammatory effect of CS-LO may due to its inhibition of NO and PGE 2 via blocking the expression of iNOS and COX-2.
TNF-, IL-6, and L-1 , a few of important proinflammatory mediators, play important roles in mediating and regulating inflammation in vitro and in vivo. TNF-, one of the cytokines produced mainly by activated macrophages, can promote inflammatory activity by regulating some adhesion molecules [23]. IL-6 can promote the differentiation of T and B lymphocytes and the release of some inflammatory cytokines [24]. IL-1 was one of the earliest expressed proinflammatory cytokines [25]. Overexpression of both IL-6 and IL-1 is considered to be an important role in the pathophysiology of rheumatoid arthritis [26]. In this study, TNF-, IL-1 , and IL-6 level, which were highly increased by LPS, were inhibited by CS-LO (Figures 1(c), 1(d), and 1(e)).
NF-B plays an important role in regulating immune system and inflammatory processes. Activated NF-B regulates the transcription of inflammatory cytokines and enzymes, such as iNOS, COX-2, TNF-, IL-1 , and IL-6 [27]. NF-Bp65, a signal for NF-B activation, played an important role in the regulation of its transcriptional capacity. Once the NF-Bp65 is activated and phosphorylated, it could promote proinflammatory cytokines [28]. Therefore, NF-B signaling pathway is considered to be a therapeutic route against inflammatory diseases. Our findings suggest that CS-LO suppressed the nuclear translocation of p65 in a concentration-dependent manner ( Figure 3). Therefore, CS-LO might regulate the transcription of COX-2, iNOS, and other inflammatory cytokines by antagonizing the transcriptional activity of NF-B.
Carr-induced paw edema could cause the peripheral release of inflammatory cytokines and mediators in tissue [29]; it is a common model to study inflammation. After administrating with Carr, the edema will rise to the high level, PGE2, TNF-, and NO will be released during 1-5 h, and the malondialdehyde (MDA) will be produced by free radical attack on the membrane [30]. Edema and MAD are important criteria to evaluate anti-inflammatory activity of natural products [31]. Our results showed that CS-LO significantly decreased the paw edema at 5 h after treatment with Carr (Figure 4(a)), the MDA level was also significantly decreased (Figure 4(b)). These findings indicate that CS-LO has a potent anti-inflammatory activity in vivo. NO, PGE 2 , and TNF-play an important role in inflammatory response [32]. Our results revealed that CS-LO decreased the NO, PGE 2 and TNF-level after treatment with Carr (Figures 4(c), 4(d), and 4(e)). We also found that CS-LO downregulated neutrophils with H&E staining (Figure 5(d)).
These results, along with the significant suppression of LPSinduced NO, PGE 2 and TNF-a by CS-LO in macrophages, suggest that the anti-inflammatory effects of CS-LO might due to its suppression of NO, PGE 2 , and TNF-synthesis in the peripheral tissues.

Conclusions
In summary, CS-LO showed potent anti-inflammatory properties. These activities may be due to the effects of minor components or synergetic effects of the components. The current study also demonstrated that CS-LO not only inhibited iNOS and COX-2 expression and the subsequent production of NO and PGE 2 but also reduced the expression of IL-1 , IL-6, and TNFin vitro and in vivo. Moreover, the inhibition of CS-LO was related to inactivation of NF-B. The results of this study indicate that the leaf essential oil of C. subavenium is a potential anti-inflammatory agent. The Conclusions section should clearly explain the main findings and implications of the work, highlighting its importance and relevance.

Data Availability
All the data used to support the finding of this study are included within the article.