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Anti-inflammatory Effect of Essential Oil from Citrus aurantium L. var. amara Engl Chun-Yan Shen,† Jian-Guo Jiang,*,† Wei Zhu,*,‡ and Qin Ou-Yang§ †

College of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong 510640, People’s Republic of China ‡ The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510120, People’s Republic of China § Wenzhou Central Hospital, Wenzhou, Zhejiang 325000, People’s Republic of China ABSTRACT: Essential oil has been popularly used as an alternative for the treatment of inflammation. The bioactivities of essential oil from blossoms of Citrus aurantium L. var. amara Engl (CAVAO) showed greater anti-inflammation potential than that of antioxidant, anticancer, and 3T3-L1 proliferation inhibition. CAVAO (250 μg/mL) significantly inhibited production of nitric oxide (NO) (99.54 ± 2.81%), interleukin-6 (IL-6) (98.11 ± 1.62%), tumor necrosis factor-α (TNF-α) (41.84 ± 1.52%), and interleukin-1β (IL-1β) (56.09 ± 2.21%) as well as their gene expression level. CAVAO also markedly decreased the expression levels of cyclooxygenase-2 (COX-2) gene and protein. Furthermore, CAVAO inhibited nuclear factor-κB (NF-κB) activation, which was justified by the inhibitory effect on NF-κB nuclear translocation, IκBα phosphorylation and degradation, and phosphorylation-dependent IκB kinase activation in RAW264.7 cells stimulated with lipopolysaccharides. CAVAO also suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38, indicating that mitogen-activated protein kinase (MAPK) signaling pathways were also blocked. The major constituents of CAVAO were characterized as linalool (64.6 ± 0.04%), α-terpineol (7.61 ± 0.03%), (R)-limonene (6.15 ± 0.04%), and linalyl acetate (5.02 ± 0.03%), which might be responsible for its observed anti-inflammation activity. It is concluded that CAVAO has great potential to be developed into a functional food for the treatment of inflammatory-associated diseases. KEYWORDS: essential oil, inflammation, RAW264.7, MAPK, NF-κB

1. INTRODUCTION A pro-inflammatory mediator at a low level was beneficial for humans; however, its excess production can induce various diseases, such as arthritis, atherosclerosis, and even cancer.1−4 During inflammatory processes, macrophages are directly involved with the inflammatory response and play a key role in regulating the innate immune responses. Mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways were involved with production of pro-inflammatory mediators, which were validated by a growing number of published data.5,6 Moreover, the MAPK signaling pathway plays a crucial role in activating the NF-κB pathway. In support, some phytochemicals were found to inhibit activation of both NF-κB and MAPK.7 Hence, the NFκB and MAPK signaling pathways have become attractive and potential targets for the treatment of inflammation. Natural products have attracted much attention, owing to few side effects.8 Essential oil, a kind of natural compound used as an alternative for treatment of various diseases, has appeared to exhibit particular medicinal properties, and their sedative, anti-anxiety, antidepressant, antibacterial, and antifungal effects have been confirmed by numerous published papers.9,10 Furthermore, essential oils have provided considerable potential for inflammatory treatment over the past century. Specifically, an accumulating body of evidence described the antiinflammation effect as well as the chemical composition of essential oil from Citrus.11−13 Citrus aurantium L. var. amara © 2017 American Chemical Society

Engl (CAVA) belongs to the Rutaceae family and has plenty of essential oil commonly known as neroli.14 Chemical constituents and bioactivity analyses of CAVA were frequently studied in our laboratory.15−19 However, the anti-inflammation effect of essential oil from blossoms of CAVA (CAVAO) has not been investigated. Here, this research was undertaken to determine the anti-inflammation effect of CAVAO and explore its underlying molecular mechanism. We also provided evidence that linalool, α-terpineol, (R)limonene, and linalyl acetate might be responsible for the observed activity.

2. MATERIALS AND METHODS 2.1. Chemicals. The nitric oxide (NO) assay kit, NF-κB activation−nuclear translocation assay kit, and nuclear and cytoplasmic protein extraction kit were purchased from Beyotime Biotech (Guangzhou, China). Enzyme-linked immunosorbent assay (ELISA) kits for determining interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were obtained from Cusabio Biothch (Wuhan, China). Antibodies to GAPDH, p-SAPK/JNK, pp38 MAPK, p-NF-κB p65, NF-κB p65, IκBα, p-IκBα, p-IκKα/β, and cyclooxygenase-2 (COX-2) were obtained from Cell Signaling Technology (Beverly, MA, U.S.A.). The reagents used in gas Received: Revised: Accepted: Published: 8586

June 4, 2017 September 3, 2017 September 14, 2017 September 14, 2017 DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

Article

Journal of Agricultural and Food Chemistry

Dexamethasone (DXM) (50 μg/mL) was used as a positive control during the research. 2.8.2. Quantification of Cytokines by ELISA. After incubation with CAVAO for 24 h, the supernatants were collected and centrifugated. Then, the secretion of IL-6, TNF-α, and IL-1β was determined with the commercially available ELISA kits based on the protocols of the manufacturer. 2.8.3. Reverse Transcription and Real-Time Quantitative Polymerase Chain Reaction (PCR). After incubation for 12 h with CAVAO and LPS, total RNA was extracted by Trizol reagent.23 The purity of RNA was determined at 260 and 280 nm using a Nano Drop spectrophotometer (Nano Drop Technologies, Wilmington, DE, U.S.A.). cDNA was amplified using the DyNAmo Flash SYRB Green qPCR Kit. The sequences of the primers used were as follows: inducible nitric oxide synthase (iNOS) (forward, 5′-CGGCAAACATGACTTCAGGC-3′; reverse, 5′-GCACATCAAAGCGGCCATAG3′), IL-6 (forward, 5′-TACTCGGCAAACCTAGTGCG-3′; reverse, 5′-GTGTCCCAACATTCATATTGTCAGT-3′), TNF-α (forward, 5′-GGGGATTATGGCTCAGGGTC-3′; reverse, 5′-CGAGGCTCCAGTGAATTCGG-3′), IL-1β (forward, 5′-TGAAGGGCTGCTTCCAAACCTTTGACC-3′; reverse, 5′-TGTCCATTGAGGTGGAGAGCTTTCAGC-3′), and COX-2 (forward, 5′-CAGCA AATCCTTGCTGTTCC-3′; reverse, 5′-TGGGCAAAGAATGCAAACATC-3′). 2.8.4. Immunostaining for Translocation of p65. RAW264.7 cells grown on glass coverslips were fixed with the stationary liquid for 15 min, blocked for 1 h, and then incubated overnight at 4 °C with the primary antibody NF-κB p65. The next day, the cells were incubated with the Cy3-labeled secondary antibody for 1 h and stained with 2-(4amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) solution for 5 min. It was important that cells were washed 3 times by washing buffer in each procedure. Eventually, the activation and nuclear translocation of NF-κB were measured via a laser scanning confocal microscope. 2.8.5. Western Blotting Analysis. RAW264.7 cells were scraped into cold PBS and centrifuged at 2000 rpm. The resulting cell pellets were mixed with radioimmunoprecipitation assay (RIPA) lysis buffer and lysis on ice for 40 min with vigorous vortexing.24 Eventually, the whole cell lysate were obtained after centrifugation at 12 000 rpm for 20 min. The cytoplasmic and nuclear proteins were prepared by the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech, China) based on the instructions of the manufacturer. In brief, the pellets were mixed with cytoplasmic protein extraction agent A supplemented with phenylmethylsulfonyl fluoride (PMSF) (1 mM). Then, the pellets were dispersed completely by 5 s of drastic vortexing and incubated on ice to promote lysis. After 15 min, the cytoplasmic protein extraction agent B was added, followed with 5 s of violent vortexing and 1 min of incubation on ice. Then, the cytoplasmic protein was collected after centrifugation at 12000g for 5 min. Furthermore, the remaining pellets were resuspended and mixed well in nuclear protein extraction agent pretreated with PMSF (1 mM) by 15−30 s of vortexing. Then, the samples were incubated for another 30 min together with 15−30 times of vigorous vortexing. Finally, the supernatants containing the nuclear protein were obtained following centrifugation at 12000g for 10 min. Then, 40 μg of proteins was separated by electrophoresis through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS− PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were then incubated with blocking solution [5% bovine serum albumin (BSA) in 1× Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST)] for 1 h with gentle shaking to block non-specific binding. Thereafter, the PVDF membranes were incubated with various primary antibodies (phospho-JNK, phospho-p38, phospho-p65, COX-2, IκBα, phosphoIκBα, phospho-IκKα/β, and NF-κB p65) overnight at 4 °C at 1:1000 dilution (v/v) in 5% BSA, followed by incubation with horseradishperoxidase-linked secondary antibody at 1:2000 (v/v) for 1 h. Each step above was followed by washing 3 times with 1× TBST for 10 min each. Finally, the blots were probed using enhanced chemilumines-

chromatography−mass spectrometry (GC−MS) experiments were of high-performance liquid chromatography (HPLC) grade. 2.2. Plant Materials. Dried blossoms of CAVA were obtained from Qingping traditional Chinese medicine market in Guangzhou, China. The plant material was washed and dried at 60 °C in a hot-air oven for 24 h. Then, the materials were smashed with a lab mill and passed through a 60-mesh sieve to obtain fine power. 2.3. Extraction of Essential Oil. Initially, 100 g of dried and ground material was weighed and soaked in 2000 mL of distilled water for 1 h and extracted by steam distillation for another 6 h. After natural cooling for 30 min, the separate layer on the water was extracted 4 times by an equal volume of diethyl ether.20 Then, the pale yellow essential oil (CAVAO) was collected, decanted into brown glass bottles, and stored at 4 °C for future biological and analytical tests. Extraction was performed in triplicate. 2.4. GC−MS Analysis. CAVAO was analyzed by GC−MS based on the published protocol.20 Briefly, GC−MS analysis was performed on an Agilent 6890N gas chromatograph equipped with a HP-5MS column (30 m × 0.25 mm, 0.25 μm film thickness) and an Agilent model 5975 insert mass spectrometer detector. The initial column temperature varied from 50 °C (held for 8 min) to 130 °C at 8 °C/ min, then raised to 200 °C at 5 °C/min, and then increased to 280 °C (held for 4 min) at 15 °C/min. The injector port temperature was held at 280 °C. The relative proportion of each compound identified from CAVAO was calculated according to the peak area normalization method, and the result was expressed as means of triplicate experiments. 2.5. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2′Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) Radical Scavenging Activity of CAVAO. The antioxidant effect of CAVAO was determined by DPPH and ABTS methods based on previous reports,21,22 with slight modifications. When the DPPH radical scavenging activity was determined, CAVAO of different concentrations ranging from 50 to 800 μg/mL was mixed with DPPH solution (150 μmol/L). After 30 min, optical density at 517 nm (OD517) was measured. Similarly, the ABTS radical scavenging activity was also evaluated at 734 nm with vitamin C used as a positive control. The inhibitory percentage of DPPH as well as ABTS was calculated according to the following formula:

SE (%) =

(ODcontrol − ODsample ) ODcontrol

× 100%

(1)

where SE was the scavenging effect and ODsample and ODcontrol were the absorbances of DPPH (ABTS) radical solution with or without CAVAO, respectively. 2.6. Cell Culture. Human hepatocyte cells LO2, human breast cancer cells MCF-7, mouse preadipocyte cells 3T3-L1, and murine macrophages RAW264.7 were maintained at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). CAVAO was dissolved in dimethyl sulfoxide (DMSO) and filtered through a 0.25 μm filter membrane. During the experiment, the samples were diluted in DMEM to various working concentrations varying from 15.625 to 250 μg/mL. 2.7. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay for Cell Cytotoxicity. LO2, RAW264.7, 3T3-L1, and MCF-7 cells were incubated with or without CAVAO for 24 h in 96-well plates. Afterward, the culture media was discarded, and 200 μL of MTT [dissolved in phosphate-buffered saline (PBS) at 5 mg/mL] in DMEM was added. After 4 h, the supernate was discarded and 150 μL of DMSO was added. Finally, OD490 were measured on a microplate reader to evaluate the cell viability and proliferation inhibitory ratio. 2.8. Anti-inflammatory Activity Assay. 2.8.1. Determination of Morphology and NO Production. RAW264.7 cells were initially stimulated with CAVAO at different concentrations of 16.13, 31.25, 62.5, 125, and 250 μg/mL for 2 h. Then, lipopolysaccharide (LPS) (1 μg/mL) was added and incubated for another 24 h. The representative micrographs of the macrophages were obtained by invert microscopes. Moreover, the accumulation amount of NO was determined with Griess reagent as previously described, with slight modifications.20 8587

DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

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Journal of Agricultural and Food Chemistry

Figure 1. (A) Total ion chromatograms of GC−MS of CAVAO. The retention times of identified compounds (relative percent contents of >1%) were showed in panel A. Retention times of 9.897, 11.884, 13.272, 13.704, 14.038, 15.811, 16.098, 17.412, 19.476, 19.883, 19.887, 20.011, 20.564, 21.262, and 23.852 min represented (1S)-(1)-β-pinene, (R)-limonene, linalool oxide, cis-5-ethenyltetrahydro-α,α-5-trimethyl-2-furanmethanol, linalool, terpinen-4-ol, α-terpineol, linalyl acetate, neryl propanoate, geranyl acetate, nerol acetate, (E,E)-farnesyl acetate, l-caryophyllene, (E)-βfarnesene, and 4,5-epoxy-4,11,11-trimethyl-8-methylenebicyclo(7.2.0)undecane, respectively. (B) Chemical compositions of CAVAO. cence (ECL) and autoradiographed. During the experiment, GAPDH antibody was used to normalize the relative band intensity. 2.9. Statistical Analysis. The data were obtained from at least three separate experiments and present as the mean ± standard deviation (SD). Statistical analysis was carried out using Statistical Package for the Social Sciences 20.0 software.

of 5-fluorouracil. CAVAO showed significant cell proliferation inhibitory effects on 3T3-L1 cells within 31.25−250 μg/mL. At 250 μg/mL, the proliferation inhibitory effect of CAVAO on 3T3-L1 cells increased sharply and reached 45.46%, indicating that CAVAO might provide new potential for weight loss. Supportedly, Haaz et al. reported that fruits of C. aurantium were often incorporated into supplements designed to aid in weight loss. The proliferation inhibitory activity of CAVAO on 3T3-L1 cells might be related to the presence of alcohols and aldehyde ketones in CAVAO according to previous reports.26,27 NO was closely related to gene expression levels of iNOS. Activation of iNOS could produce a lot of NO by acting on amino acid L-arginine. CAVAO dose-dependently inhibited NO accumulation, and the inhibition ratio surpassed that of the positive control DXM (50 μg/mL) at 250 μg/mL. RAW264.7 cells stimulated with LPS increased in size and became irregular in shape. RAW264.7 cells in the CAVAO-treated group displayed relatively smooth surfaces compared to those in the LPS-stimulated group (Figure 3). In conclusion, all of these results indicated that CAVAO probably exerts a better antiinflammation effect than that of weight loss and antitumor. Therefore, a further study was conducted to gain insight into the underlying mechanisms. 3.3. Effect of CAVAO on LPS-Induced iNOS, IL-6, TNFα, IL-1β, and COX-2. As shown in panels A−C of Figure 4, CAVAO treatment potently suppressed secretion of IL-6, TNFα, and IL-1β in LPS-induced RAW264.7 cells, with a maximum inhibitory ratio that exceeded that of DXM at 250 μg/mL. At the same time, the mRNA expressions of IL-6 (Figure 4E), TNF-α (Figure 4F), and IL-1β (Figure 4G) were also decreased after administrated with CAVAO. Obviously, CAVAO showed a slightly lower inhibitory effect on TNF-α gene expression than IL-6 and IL-1β genes. One of the possible explanations would be that TNF-α might act through different pathways.28 Figure 2D demonstrated that CAVAO could dramatically decrease the mRNA expression of iNOS and the anti-inflammation effect was much superior to DXM, corresponding to the downregulation of NO production. A growing number of previous studies also provided evidence for this,20,29 suggesting that NO and iNOS might serve as targets for novel therapeutic options. Additionally, LPS treatment led to a significant increase of COX-2 mRNA compared to the normal control group; however, this increase was dramatically inhibited when CAVAO (62.5−250 μg/mL) was administrated (Figure 4H).

3. RESULTS 3.1. Comparative Analysis of Essential Oil Compounds. CAVAO extracted from blossoms of CAVA by steam distillation was yellowish with a pleasant odor, and the yield was 0.18% (w/w) based on dried sample powder. As shown in Figure 1A, there were many individual peaks in the total ion chromatograms of GC−MS of CAVAO. A total of 31 volatiles, representing 98.821% of the total chromatographical material of CAVAO, were identified (Table 1). The prominent components of CAVAO were characterized as linalool (64.6 ± 0.04%), α-terpineol (7.61 ± 0.03%), (R)-limonene (6.15 ± 0.04%), and linalyl acetate (5.02 ± 0.03%), respectively. Of note, linalool comprised most of the neroli composition, in accordance with the published data.25 Furthermore, Figure 1B visually illustrated the varieties as well as the percent contents of CAVAO. Alcohols were the most abundant compounds, representing 75.598% of the total volatile emission in CAVAO, followed by alkenes (12.269%), esters (8.645%), acids (1.807%), aromatic compounds (0.277%), alkanes (0.088%), and aldehyde ketones (0.024%) (Figure 1C). Specifically, alcohols were mostly represented by the appreciability of linalool (64.6 ± 0.04%), α-terpineol (7.61 ± 0.03%), and linalool oxide (1.73 ± 0.02%). As a matter of fact, the species of identified compounds and their percent contents in this study were a little different from the previous reports.11,25 One of the possible explanations would be that these qualitative and quantitative differences in oil compositions probably depended upon various factors, such as geographical location, climate, and season. 3.2. Determination of the Antioxidant Activity, Aytotoxicity on 3T3-L1 and MCF-7 Cells, and Inhibitory Effects on LPS-Induced NO Production. Antioxidant activity, weight loss, anticancer activity, and anti-inflammation effects of CAVAO were determined in vitro. CAVAO did not show profound scavenging effects on DPPH (Figure 2A) and ABTS (Figure 2B) radicals. At 15.625−250 μg/mL, CAVAO showed no cytotoxicity on LO2 and RAW264.7 cells (Figure 2C). Simultaneously, the proliferation inhibitory rate of CAVAO on MCF-7 cells (Figure 2D) was far less than that 8588

DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

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Journal of Agricultural and Food Chemistry Table 1. Chemical Composition of Essential Oils Extracted from CAVA Identified by GC−MS retention time (min)

CAS Registry Number

12.655

005794-03-6

molecular formula

compound

relative percent content (%)a

Alkane

11.773

000535-77-3

7.834 9.897 10.728 11.884 14.959 20.564 20.821 21.253 21.262 23.852

003779-61-1 018172-67-3 000127-91-3 005989-27-5 003338-55-4 000087-44-5 000502-61-4 026560-14-5 018794-84-8 001139-30-6

13.272 13.704 14.038 15.811 16.098 17.990 18.076

001365-19-1 005989-33-3 000078-70-6 000562-74-3 000098-55-5 026532-23-0 000106-25-2

17.986

005392-40-5

17.412 19.476 19.883 19.887 30.661

000115-95-7 000105-91-9 000105-87-3 000141-12-8 006929-04-0

10.715 20.011 30.648

001846-70-4 004128-17-0 001731-88-0

D-camphene

Aromatic Compounds 1-methyl-3-(1-methylethyl)benzene Alkenes (E)-ocimene (1S)-(1)-β-pinene β-pinene (R)-limonene (Z)-13,7-dimethyl-3,6-octatriene l-caryophyllene α-farnesene (3Z,6E)-α-farnesene (E)-β-farnesene 4,5-epoxy-4,11,11-trimethyl-8- methylenebicyclo(7.2.0)undecane Alcohols linalool oxide cis-5-ethenyltetrahydro-α,α-5-trimethyl-2-furanmethanol linalool terpinen-4-ol α-terpineol cis-3,3-dimethyl-Δ1,β-cyclohexaneethanol neryl alcohol Aldehyde Ketones citral Esters linalyl acetate neryl propanoate geranyl acetate nerol acetate hexadecanoic acid, 15-methyl-, methyl ester Acids 2-nonynoic acid (E,E)-farnesyl acetate methyl tridecanoate Others α-humulene epoxide II N-isopropyl-3-phenylpropanamide N-(3-methylbutyl) acetamide

24.383 019888-34-7 24.859 056146-87-3 31.440 013434-12-3 yield of essential oils (%, w/w) detected species of compounds identified compound percentage of the total content of essential oils (%) a

C10H16

0.088 ± 0.01

C10H14

0.277 ± 0.01

C10H16 C10H16 C10H16 C10H16 C10H16 C15H24 C15H24 C15H24 C15H24 C15H24O

0.088 ± 0.02 1.88 ± 0.02 0.144 ± 0.01 6.15 ± 0.04 0.148 ± 0.01 1.35 ± 0.03 0.058 ± 0.01 0.589 ± 0.02 0.640 ± 0.03 1.76 ± 0.01

C10H18O2 C10H18O2 C10H18O C10H18O C10H18O C10H18O C10H18O

1.73 ± 0.02 1.01 ± 0.02 64.6 ± 0.04 0.514 ± 0.02 7.61 ± 0.03 0.113 ± 0.01 0.060 ± 0.01

C10H16O

0.024 ± 0.00

C12H20O2 C13H22O2 C12H20O2 C12H20O2 C18H36O2

5.02 ± 0.03 1.16 ± 0.02 1.25 ± 0.02 1.03 ± 0.02 0.184 ± 0.01

C9H14O2 C17H28O2 C14H28O2

0.63 ± 0.01 1.15 ± 0.03 0.027 ± 0.01

C15H24O C12H17NO C7H15NO

0.014 ± 0.01 0.010 ± 0.01 0.089 ± 0.01 0.18 31 99.4

Bold numbers mean relative percent contents of >1%.

with the immunofluorescence analysis. Furthermore, the data indicated that CAVAO could significantly inhibit the phosphorylation and proteolytic degradation of IκBα in macrophages. It was noteworthy that IκBα was remarkably upregulated when administrated with CAVAO varying from 62.5 to 250 μg/mL (Figure 6E). Of note, the expression levels of IκBα exceeded that of control cells. Meanwhile, phosphoIκBα was downregulated after treatment with CAVAO at 62.5 μg/mL (Figure 6F). Additionally, CAVAO markedly suppressed LPS-induced phosphorylation of IκKα/β concentration-dependently, and the expression levels of phospho-IκKα/β in the CAVAO-treated group (125 and 250 μg/mL) were lower than that in the control group (Figure 6G). Collectively, these results indicated that CAVAO could inhibited NF-κB activation. Supportedly, CT20126 was also

Indeed, the regulatory activity was nearly comparable to that of DXM. 3.4. Effect of CAVAO on LPS-Induced NF-κB Activation. The immunofluorescence analysis visually demonstrated that NF-κB p65 translocated from cytosol to the nucleus compared to the normal control cells (Figure 5). However, CAVAO could markedly suppress this condition, suggesting that CAVAO might exert anti-inflammatory activity via inhibiting NF-κB activation. The results were further confirmed by western blot analysis. CAVAO dramatically suppressed LPSinduced activation of phospho-p65 (Figure 6B) in a dosedependent manner, whereas p65 (Figure 6A) was not significantly affected. Simultaneously, CAVAO administration enhanced the cytosolic p65 subunit level (Figure 6C) and decreased the nuclear p65 (Figure 6D) level, in accordance 8589

DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

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Journal of Agricultural and Food Chemistry

Figure 2. Antioxidant activity of CAVAO determined by the (A) DPPH free radical scavenging asssy and (B) ABTS free radical scavenging asssy, cytotoxicity of CAVAO toward (C) LO2 and RAW264.7 cells, (D) MCF-7 cells, and (E) 3T3-L1 cells, and (F) NO inhibition effect of CAVAO on LPS-induced RAW264.7 cells.

inhibit COX-2 protein expression, thereby downregulating the expression of COX-2 mRNA.

4. DISCUSSION NF-κB, generally existing in a heterodimeric inactive form, could be activated and translocated from the cytoplasm to the nucleus as a result of the serine phosphorylation and subsequent proteolytic degradation of IκB by activating IκB kinase (IκK), which is responsible for IκB phosphorylation. Thus, NF-κB is liberated, translocated from cytoplasm into nuclei, and bound to promoters of target genes, thereby promoting the expression of various inflammatory mediators. For example, the NF-κB binding element at −85 base pairs (bp) in the murine iNOS promoter is a primary factor associated closely with iNOS expression and NO production, which could explain the result in section 3.2. Our results showed that CAVAO could significantly suppress NF-κB p65 translocation by inhibiting phosphorylation and proteolytic degradation of IκBα through regulating IκK activation and activity. The MAPK signaling pathway is well-known to play central roles in signal transduction during inflammatory response. For instance, the JNK pathway was confirmed to be related to accumulation of NO, IL-6, and TNF-α.34 Also, the p38 pathway was assumed to regulate the expression of the TNF-α gene.35 Figure 6 demonstrated that CAVAO dose-dependently inhibited phosphorylation of JNK and p38, indicating that the prohibitive effect of CAVAO on pro-inflammation mediators might be through MAPK pathways. In accordance with our results, some other studies also proved to act similarly.33,36 In fact, MAPK is an upstream activator of NF-κB and could trigger NF-κB activation. In this study, CAVAO not only suppressed LPS-induced nuclear translocation of NF-κB but also inhibited phosphorylation of JNK and p38 (Figure 7), suggesting that CAVAO might inhibit NF-κB and MAPK activation, thereby regulating the pro-inflammatory mediators. As a matter of fact, CAVAO exhibited a higher inhibitory effect on the expression

Figure 3. Pictures of RAW264.7 cells by an invert microscope after treated with different samples.

reported to exert anti-inflammatory effects through inhibiting NF-κB activation.30 3.5. Effect of CAVAO on LPS-Induced MAPK Phosphorylation and COX-2 Expression. Recently, accumulating evidence proved that inhibition of MAPK phosphorylation could inhibit inflammation and relieve inflammatory conditions.31−33 As demonstrated in Figure 6H, phosphorylation of c-Jun N-terminal kinase (JNK) was prominently upregulated when treated with LPS. However, this enhancement was significantly decreased when pretreated with CAVAO for 3 h. Meanwhile, CAVAO also inhibited phosphorylation of p38 dose-dependently when the concentration varied from 62.5 to 250 μg/mL (Figure 6I). Specifically, the expression level of phospho-p38 in macrophages pretreated with CAVAO at 250 μg/mL was close to that of the control cells without samples. In summary, the experimental data suggested that CAVAO might involve the inflammation process through MAPK signaling pathways, which were mediated by inhibiting phosphorylation of JNK and p38. Additionally, western blot analysis demonstrated that the inhibitory effect of CAVAO on COX-2 protein expression (Figure 6J) was consistent with that on COX-2 mRNA expression, indicating that CAVAO might 8590

DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

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Journal of Agricultural and Food Chemistry

Figure 4. Effect of CAVAO on LPS-induced secretion of (A) IL-6, (B) TNF-α, and (C) IL-1β an deffect of CAVAO on LPS-induced mRNA expression levels of (D) iNOS, (E) IL-6, (F) TNF-α, (G) IL-1β, and (H) COX-2. All experiments were run in triplicate, and data showed the mean ± SD values. (∗) p < 0.05 and (∗∗) p < 0.01 compared to the LPS-treated group, while (##) p < 0.01 compared to the control group.

of inflammation-associated genes than that on NF-κB and MAPK. One possible reason would be that the proinflammatory mediators could also be regulated by other pathways.37,38 As demonstrated in section 3.1, linalool (64.6 ± 0.04%), αterpineol (7.61 ± 0.03%), (R)-limonene (6.15 ± 0.04%), and linalyl acetate (5.02 ± 0.03%) composed 83.4% of the constituents. Few study on essential oil reported such a high percentage; therefore, the phytochemical results indicated that the four compounds might represent CAVAO and be responsible for its observed anti-inflammation activity. Held et al. identified significant amounts of limonene, linalool, and α-

terpineol in the oral buccal cells treated with volatile compounds of orange juice using GC−MS and found that αterpineol effectively inhibited IL-6 formation, while linalool and limonene showed no or even stimulating effects on IL-6 formation.39 These experimental data suggested that αterpineol (3144 μg/L) might be the active principle for the anti-inflammatory effect of aqueous distillate (AD) by inhibiting IL-6 formation. The percent recovery of α-terpineol in the exponate of the cells (cell culture medium + AD), the air space of the cell culture flask, and the cell lysate was determined to be 88%, and that in the lysate of the cells was 0.4%. Simultaneously, the anti-inflammatory effect of linalool was 8591

DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

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Journal of Agricultural and Food Chemistry

also proven by cigarette-induced acute lung inflammation and LPS-stimualted BV2 microglia cells.40,41 Peana et al. further justified that the samples containing linalool and linalyl acetate were potential anti-inflammatory agents.42 Reportedly, most linalool and linalyl acetate disappeared quickly from the blood, and their biological half-lives were approximately 14 min.43 In fact, published reports showed that only 3% of linalool was detected in tissues after 72 h of oral administration.44,45 Additionally, limonene has attracted much attention and been recognized as a potent anti-inflammatory compound based on the study on mouses, rats, and elderly humans.46,47 The Rlimonene metabolism and elimination kinetics in a human were conducted by Schmidt et al., and they found that the metabolite amount excreted via urine was about 38.8% of the orally administered dose after 24 h.48 Although the influence of the key molecules on the bioactivity was not studied, published reports found that various chemical compounds, even for those presented at low concentrations, might produce synergistic or additive effects.49 It was possible that the anti-inflammation

Figure 5. Analysis of nuclear translocation of NF-κB determined by immunofluorescence analysis with a laser scanning confocal microscope.

Figure 6. Effects of CAVAO on (A) p65, (B) phospho-p65, (C) cytosol p65, (D) nucleus p65, (E) IκBα, (F) phospho-IκBα, (G) phospho-IκKα/β, (H) phospho-JNK, (I) phospho-p38, and (J) COX-2. All experiments were run in triplicate, and data showed the mean ± SD values. (∗) p < 0.05 and (∗∗) p < 0.01 compared to the LPS-treated group, while (##) p < 0.01 compared to the control group. 8592

DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

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REFERENCES

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Figure 7. Possible molecular mechanisms of the anti-inflammation activity of CAVAO in RAW264.7 cells.

activity of CAVAO was a result of a complex interaction among its various chemical compounds, which might produce synergistic or additive effects, even for those presented at low concentrations. In summary, CAVAO might be a potential candidate for developing new therapeutic supplements. CAVAO at 250 μg/ mL showed greater anti-inflammation potential compared to many other crude extracts or even compounds, such as polysaccharides from Smilax glabra at 400 μg/mL34 and tiliroside at 100 μg/mL.29 This study was a systematic report about bioactivities of essential oil extracted from blossoms of CAVA, and the results paved the way for prospective clinical trials to evaluate the potential anti-inflammation effects of CAVAO. However, we acknowledge that this study is in the very earliest of stages, and more studies, such as bioavailability and metabolic effects in vivo, are urgently needed for clinical treatment in long-term use.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-20-87113849. Fax: +86-20-87113843. E-mail: [email protected]. *Telephone: +86-20-39318571. Fax: +86-20-39318571. E-mail: [email protected]. ORCID

Jian-Guo Jiang: 0000-0002-3361-6149 Funding

This project was supported by the Science and Technology Project of Guangzhou City (201604020150), the Guangdong Science and Technology Project (2013B090700015), the Scientific Research Projects of the State Administration of Traditional Chinese Medicine (JDZX2015205), the Guangdong Provincial Science and Technology Department Project (2015B020211013), the Science and Technology Project of Wenzhou City (Y20150011), and the Guangdong Provincial Hospital of Chinese Medicine Science and Technology Research Program (YN2016MJ02). Notes

The authors declare no competing financial interest. 8593

DOI: 10.1021/acs.jafc.7b02586 J. Agric. Food Chem. 2017, 65, 8586−8594

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