Topical Anti-Inflammatory Activity of Essential Oils of Alpinia calcarata Rosc., Its Main Constituents, and Possible Mechanism of Action

This study aimed at investigating the anti-inflammatory potential of essential oil from rhizome and leaf of Alpinia calcarata Rosc. (ACEO) with the focus of its topical anti-inflammatory activity along with its dominant compounds 1,8-cineole and α-terpineol using mouse ear edema model. ACEOs were analyzed by GC-MS. The anti-inflammatory activity was determined by studying the inhibition of overproduction of proinflammatory mediators—nitric oxide, reactive oxygen species, prostaglandins, cyclooxygenases, and cytokines induced by lipopolysaccharides in murine macrophages. Topical anti-inflammatory and antinociceptive activity was studied by 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin inflammation and formalin-induced pain model in mice, respectively. Rhizome oil has 1,8-cineole (31.08%), α-terpineol (10.31%), and fenchyl acetate (10.73%) as major compounds whereas the ACEO from leaves has 1,8-cineole (38.45%), a-terpineol (11.62%), and camphor (10%). ACEOs reduced the production of inflammatory mediators in vitro in a concentration-dependent manner. Further, ACEO and its major compounds reduced ear thickness, weight, myeloperoxidase, and cytokines significantly (p < 0.01) in mouse ear. Dose-dependent reduction in flinching and licking in both the phases of pain sensation concludes the topical analgesic effect. Our findings suggest the potency of topical use of ACEOs for inflammatory disease conditions.


Introduction
Inflammation is a defending process exhibited by organisms against noxious stimuli, marked by an intensified blood influx to the affected tissue resulting in pain, heat, swelling, redness, and loss of function of the affected part [1,2]. Referred commonly as "double-edged sword," it aids in the elimination of pathogens, whereas uncontrolled inflammation could lead to tissue injury and neoplastic transformation [3]. Further, inflammation-related acute and chronic diseases are accompanied by pain which subjugates the quality of life and overall productivity [4]. Macrophages, the remarkably plastic cells of the immune system, get activated in the inflammatory process, thereby producing proinflammatory mediators such as nitric oxide (NO), PGE2 (prostaglandin E2), COX 1 and 2 (cyclooxygenase 1 and 2), reactive oxygen species (ROS), and cytokines [5]. Skin acts as the primary interface between the body and the external environment and provides the first line of defence against disease-causing pathogens and traumatic injury [6]. In addition, as a physical barrier [7], the skin has many active immune defence mechanisms. A breach in the immunological balance can head to acute and chronic inflammatory skin diseases such as psoriasis and allergic contact dermatitis [8]. In this condition, topical treatments of skin diseases have combined benefits that include simplicity in application, escaping of hepatic first-pass metabolism, attaining maximum efficacy with less drug dosage, easy termination of drug if needed, site-specific drug delivery, high adherence, and risks associated with oral or intravenous administration [9,10]. Further, topical anti-inflammatory agents can inhibit the variety of factors and mediators of inflammation such as expression of cytokines, growth factors, adhesion molecules, nuclear factor-κB (NF-κB), nitric oxide, and prostanoids [11]. TPA-induced skin inflammation is a widely used model to study the antiinflammatory effects of natural and chemically synthesized drugs. High levels of inflammatory cytokines and reactive oxygen species are proposed to contribute to the pathophysiological mechanisms associated with TPA-induced cutaneous inflammation [12]. Conventional nonsteroidal anti-inflammatory drugs (NSAIDs) ameliorate the inflammation by suppressing the mediators and also act as local analgesics [13]. However, evidences suggest that the longterm use of NSAIDs may induce gastrointestinal ulcers, bleeding, and renal disorders leading to the exploration of less or noninvasive therapeutics which could aid in the treatment strategy of the acute and chronic inflammatory diseases [14].
Alpinia calcarata Rosc. (Zingiberaceae) is a widespread perennial plant throughout the tropical and subtropical Asian countries including Sri Lanka, India, Bangladesh, ailand, and Malaysia [15,16]. Rhizomes have been commonly used in Sri Lankan and Indian traditional medicine to treat chronic inflammatory diseases such as rheumatism and asthma [17]. Studies on the hot water, ethanolic extract, and oil extract of rhizome exhibited potent anti-inflammatory activity in carrageenan-induced mice models [18][19][20]. Previous findings reported that extracts of AC had antimicrobial, antifungal, antihelminthic, antinociceptive, antioxidant, aphrodisiac, gastroprotective, and antidiabetic properties [21]. Earlier, researchers have reported the chemical composition of ACEO grown in Sri Lanka to be rich in oxygenated monoterpenes with 1,8cineole as the major constituent of rhizome and leaf EOs [22]. But this study lacks to give the detailed profile of volatile constituents from flowering AC grown in Sri Lanka. Similar supporting reports have been documented with ACEOs from germplasms in South India [23][24][25][26]. Further, the main constituents 1,8-cineole (CIN) and α-terpineol (TPN) have been known to act as anti-inflammatory agents in vivo [27,28]; its topical anti-inflammatory effect and mechanism of action for skin diseases such as atopic dermatitis were never reported. Taking this into account, we postulated that ACEO which is rich in monoterpenes like 1,8-cineole and α-terpineol could be effective in preventing TPA-induced acute skin inflammation in mice and inhibit inflammatory mediators in vitro. With the existing knowledge from literatures, this is the first study that reports the detailed profile of volatile constituents, topical anti-inflammatory activity, and in vitro mechanism of action of AC. To test this possibility, we have studied the effects of ACEO and main constituents on the TPA-induced cutaneous inflammation. In order to determine ACEOs mechanism in vitro, RAW 264.7 cells induced with LPS have been used to measure the NO, ROS, cytokines, COX, and PGE2. In addition, the cytotoxicity of the biologically active ACEOs was also evaluated on macrophages, intestinal epithelial cells, human hepatocytes, and keratinocytes in order to assess the effect if targeted for oral or topical application and further gauge the therapeutic edge over the existing drugs.

Extraction of ACEOs.
e whole plant was washed, and the rhizome and leaves were chopped separately. Each part (450 g) was separately hydrodistilled for 4 h using 500 mL distilled water in a Clevenger-type apparatus to obtain the essential oils. After decanting, oil samples were dried with anhydrous Na 2 SO 4 and stored at 4°C prior to analysis. e 2 Evidence-Based Complementary and Alternative Medicine percentage yield of oil was calculated as the ratio of weight of the oil to the weight of the fresh plant part separately. e relative amount of individual components of the total oil is expressed as percentage peak area relative to total peak area. Qualitative identification of the different constituents was performed by comparison of their relative retention times and mass spectra with those of authentic reference compounds, or by retention indices (RI) and mass spectra. Compound identification was done by comparing the National Institute of Standards and Technology (NIST 2014) library data of the mass spectra peaks with those reported in the literature.

Measurement of Cytotoxicity and Proliferative Index (PI).
e cytotoxic effects of the ACEOs were carried out on RAW 264.7, HepG2, IEC-6, and HaCaT cells through MTT assay [29] for cytotoxicity and proliferative index by the same procedure. A total of 5 × 10 4 cells/well were seeded on to 96well plates, and upon reaching the desired confluence, the cells were incubated with three different concentrations of ACEOs (0.5, 5, and 50 μg/mL) in complete culture medium, not exceeding 0.5% DMSO in content for 24 h at 37°C and 5% CO 2 . Incubation of cells with culture medium containing DMSO at a final concentration of 0.5% (v/v) was used as a negative control. e absorbance of developed formazan in the treated cells was quantified at 595 nm in a microplate reader against the DMSO (SpectraMax 384 Plus, Molecular Devices, USA). e proliferative index was carried out with a similar protocol, except the element that the cells were cultured in serum-free medium at the concentrations of 0.5, 5, and 50 μg/mL. e PI was calculated as percentage inhibition of proliferation by calculating the absorbance difference between untreated cells and treated cells.

Nitric Oxide and iNOS Production Inhibition and
Nitrite Scavenging Potential. RAW 264.7 cells were treated with LPS (1 μg/mL) in the presence of various concentrations of ACEOs 0.5, 5, and 50 μg/mL and compounds (0.32-1.25 μg/mL) for 4 h followed by removal of culture supernatant containing ACEO and added LPS containing culture media. After 20 h, the concentration of nitrite, the stable product of NO, was quantified in the culture supernatant by Griess reagents (1% sulfanilamide and 0.1% NED.2HCl) as described previously [30]. e amount of nitrite in the sample was calculated from a sodium nitrite standard curve, and the absorbance was measured by Synergy HTX multimode reader (BioTek Instruments, United States). In an attempt to determine the involvement of iNOS in NO production inhibition by ACEOs, RAW 264.7 cells were induced by LPS for 12 h prior to treatment with ACEOs for 24 h. In vitro stimulation of cells to an inflammatory state prior to treatment with drug enables the synthesis of intracellular iNOS and accumulation of high levels with corresponding enhanced synthesis and secretion of NO [31]. L-NMMA was used as a specific inhibitor of iNOS enzyme activity (positive control). e supernatants were removed and assayed for nitrite using the Griess assay as described above. In a separate experiment, the free radical nitrite scavenging ability of ACEOs was estimated by generating a NO production system with SNP (10 mM) and phosphate buffer (pH 7.4), followed by the addition of Griess reagent, and the absorbance was measured. PTIO, a synthetic nitrite scavenger, was used as a positive control.

Measurement of Intracellular ROS Production.
e inhibition of intracellular ROS production by ACEOs was quantified through chemiluminescence as described by Koko et al. [32]. Briefly, RAW 264.7 cells (1 × 10 5 cells/well) were suspended in HBSS with Ca 2+ and Mg 2+ (pH 7.4) and treated with varying concentrations of ACEO (1.56-50 μg/ mL) followed by incubation at 37°C for 30 min in the thermostated chamber of the BioTek SynergyTM multimode reader. e production of ROS was initiated by the addition of opsonized zymosan A followed by 25 μL luminol, and the volume was adjusted to 200 μL with HBSS ++ . e results were monitored as relative luminescence units (RLUs) with peak and total integral values set with repeated scans at 30 s intervals for 1 h.

Superoxide Radical Scavenging
Activity. Superoxide anion scavenging activity was measured in sodium phosphate buffer (100 mM, pH � 7.4) containing NBT solution (150 μM), NADH solution (468 μM), and different concentrations (1.56-50 μg/mL) of ACEOs. e reaction was started with the addition of PMS (60 μM) to the mixture followed by incubation at 25°C for 5 min and measurement of the absorbance at 560 nm. Compared with the optical density (OD) with no test sample added, the reduction of the absorbance was quantified as the superoxide scavenging activity.

Measurement of Cytokine
Production. Secreted cytokine levels were evaluated by incubating RAW 264.7 cells induced with 1 µg/mL of LPS and treated with ACEOs at 0.5, 5, and 50 μg/mL for 4 h. e modulatory activity of ACEO Evidence-Based Complementary and Alternative Medicine on the LPS-induced production of TNF-α, IL-1β, and IL-6 was quantified through EIA (BD, USA) using the culture supernatants of RAW 264.7 cells collected after 24 h of incubation. Dexamethasone at 10 μM was used as the positive control. 2.7.5. Measurement of PGE2 Levels. RAW 264.7 cells were induced with LPS 1 μg/mL together with ACEOs at 0.5, 5, and 50 μg/mL for 4 h. After 4 h, ACEO-containing media was removed and replaced with LPS-containing media and incubated for 24 h. After 24 h, culture supernatant was collected for ELISA quantification of PGE2 using PGE2 assay kits (ParameterTM; R&D Systems, MN, USA). e PGE2 standard and the RD5-39 in the kits were used to construct a standard curve. Culture medium (100 μL) was mixed with 50 μL of primary antibody solution and PGE2 conjugate and incubated for 2 h at room temperature with continuous shaking. Wells were then washed using 400 μL of washing buffer followed by addition of colour reagent (200 μL), and 30 min later, stop solution (50 μL) was added. Absorbance was measured at 450/570 nm using a BioTek Synergy HTX multimode reader. Indomethacin was used as the positive control.

In Vitro COX Inhibition Assay.
e efficacy of ACEOs to inhibit ovine COX-1 and COX-2 was determined using an enzyme immunoassay (EIA) kit (catalog no. 560101; Cayman Chemical Co., Ann Arbor, MI, USA). COX catalyzes the first step in the biosynthesis of arachidonic acid (AA) to PGH 2 . PGF 2α , produced from PGH 2 by reduction with stannous chloride, was measured by EIA (ACE ™ competitive EIA, Cayman Chemical, Ann Arbor, MI, USA). Briefly, to a series of supplied reaction buffer solutions (160 μL 0.1 M Tris-HCl (pH 8.0) containing 5 mM EDTA and 2 mM phenol) with either COX-1 or COX-2 (10 μL) enzyme in the presence of heme (10 μL), 10 μL of various concentrations of ACEO (0.5, 5, and 50 μg/mL) were added. ese solutions were incubated for 5 min at 37°C, and subsequently, 10 μL AA solution (100 μM) was added. Further, it was incubated for 2 min at 37°C. e COX reaction was stopped by the addition of 30 μL of stannous chloride and incubated for 5 min at room temperature. Prostaglandins, PGF 2α , produced were quantified by ELISA. is assay is based on the competition between PGs and a PG-acetylcholinesterase conjugate (PG tracer) for a limited amount of PG antiserum. Standard and samples were mixed with PG screening AChe tracer and PG antiserum and was incubated for 18 h at room temperature. After incubation, the plate was washed to remove any unbound reagent, followed by addition of Ellman's reagent (200 µL), and the mixture was incubated for 60 min at room temperature (until the absorbance of Bowell is in the range of 0.3-1.0 A.U.). e product of this enzymatic reaction develops a yellow colour that absorbs at 412 nm. e intensity of this colour is proportional to the amount of PG tracer bound to the well, which is inversely proportional to the amount of PGs present in the sample. Percent inhibition was calculated by the comparison of the compounds treated to the various control incubations.

Assay for Membrane Stabilization.
Fresh whole human blood (2 mL) was collected from a healthy volunteer and was washed three times with normal saline and constituted as 40% v/v suspension with normal saline as described by Sadique et al. [33]. e reaction mixture consisted of ACEOs (1.56-50 μg/mL) or dexamethasone (10 μM) and 20 μL of 40% RBCs suspension, considering only RBCs as controls. e reaction mixture (in triplicate) after incubation in a water bath (54°C for 25 min) was centrifuged at 2500 rpm for 5 min, and the absorbance of the supernatants was taken at 560 nm. Percent membrane stabilization activity was derived mathematically. e ethical approval for collection of blood from human donors was obtained from Research Ethics Committee, Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka.

Topical
Anti-Inflammatory Activity 2.8.1. TPA-Induced Skin Inflammation. Acute inflammation in the ear pinna was induced by instilling 20 μL of TPA dissolved in acetone (2.5 μg/ear). Mice were divided into 9 groups with 6 animals per group. TPA (2.5 μg/ear) dissolved in 20 μL of acetone was applied to the inner and outer surfaces of mouse ears with the aid of a micropipette [34]. Treatments, ACEOs (5.0, 1.0, and 0.2%), CIN (2.5, 0.5, and 0.1%), and TPN (10, 2, and 0.4%) or indomethacin (0.5 mg/ ear) was applied after the TPA induction. Four h later, the thickness of the ear was measured using a digital screw gauge. Six h after the treatment, the animals were euthanized for the collection of tissue. Ear biopsies (5 mm diameter punches) were weighed, homogenized in PBS (pH 7.5) with 1 mM EDTA, and centrifuged (10,000 ×g for 15 min) for the collection of supernatant which were used for the quantification of various cytokines.

Histopathological Analysis of Mouse Ear Tissue.
For the assessment of skin inflammation, biopsies from control and treated ears of mice in each treatment group were collected and fixed in 4% formaldehyde (0.1 M phosphate buffer, pH 7.4). Subsequently, the tissues were dehydrated, blocked in paraffin, and serially sliced at a thickness of 5.0 μM using a microtome (Leica Microsystems, USA). e sections were stained with hematoxylin-eosin (H & E), and a representative section from each group of animals was selected to show the histopathological changes. e selected sections were analyzed by light microscopy (Leica Microsystems, USA), and the images were captured at 10x and 40x magnifications. e measurements of ear thickness and epidermal thickness (μm) were acquired by the Image Processing software of Leica Microsystems.

Assay for Myeloperoxidase Enzyme Activity in Ear
Tissue. MPO activity was quantified according to the method proposed by Pulli et al. [35]. Briefly, the homogenate was added with a mixture containing 80 mM·PBS (pH 5.4), 0.22 M·PBS (pH 5.4), and 0.017% hydrogen peroxide. e reaction was initiated by the addition of 20 μL of 18.4 mM TMB in dimethylsulphoxide. e plate was incubated at 37°C for 4 min followed by the addition of 1.46 M sodium acetate (pH 3.0) to stop the reaction. e absorbance at 620 nm was measured using a plate reader to determine the enzyme activity. MPO activity was expressed as absorbance of TPA-treated ear tissue homogenate ÷ absorbance of ACEO-treated ear tissue homogenate.

Formalin-Induced Nociception and Edema in Mice.
e procedure described by Lee and Jeong [36] was followed. Briefly, nociception was induced by injecting 20 μL of 2.5% formalin in 0.9% saline in the subplantar region of the right hind paw. Mice (n � 6, per group) were pretreated topically with ACEOs (5%, 1%, and 0.25%), CIN (2.5, 0.5, and 0.1%), TPN (10, 2, and 0.4%), 1% diclofenac cream, and vehicle for 1 h prior to injecting formalin. ese mice were individually placed in a transparent glass chamber for observation. e amount of time spent licking and flinching of the injected paw was indicative of pain. e number of flinches and lickings after injection of formalin was counted during 0 to 5 min (early phase) and 20 to 30 min (late phase). Antinociception was considered as a statistically substantial reduction in the time spent in licking and flinching of the injected paw in comparison with the control group. Edema was estimated by measuring the paw volume before and after 4 h of formalin injection using plethysmometer (IITC, Life Scientific Instruments, Woodland Hills, CA, USA). e percentage reduction in paw volume by ACEOs was calculated in comparison with the formalin-treated group.

Statistical Analyses.
e raw data were analyzed by ttests and one-way ANOVA (analysis of variance) followed by Tukey's test and Dunnett's comparison test, where applicable using GraphPad Prism version 5.0 (GraphPad Prism Software Inc., San Diego, CA, USA). Differences were considered to be statistically significant when p < 0.05.

Analysis of Essential Oil.
e yield of essential oil from rhizome and leaf was 9 and 12 mL/kg, respectively. Results are the summary of three batches of analysis. Essential oils are complex mixtures consisting of various compounds. Each of these compounds contributes to the beneficial or adverse activity of the essential oil. For this reason, it is necessary to elucidate the complete composition of an essential oil when investigating the viability of a specific application [37]. GC-MS equipped with a capillary column is the most popular technique used to analyze the chemical ingredients of an essential oil. e list of compounds in ACEO from rhizome and leaf is presented in Table 1 and Figures 1(a) and 1(b). Previous study on the volatile oils of AC grown in Sri Lanka reports 3.60% and 0.42% of oil yield from dry rhizome and fresh leaves, respectively [22]. Earlier reports of rhizome EO from germplasms collected from India showed variation ranging from 0.29% to 0.96% in rhizome and 0.26% to 0.69% in aerial leafy shoots on a dry weight basis [38]. All the previous studies were done on either dry or fresh rhizome and aerial parts, and the information on the harvest stage was not mentioned. In our study, oils were collected from flowering plants. Fresh rhizome constituted mainly of oxygenated monoterpenes (71.28%), followed by monoterpenes (9.64%), sesquiterpenes (5.4%), and oxygenated sesquiterpenes (0.15%). e major compounds identified were 1,8-cineole (38.45%), α-terpineol (11.62%), and fenchyl acetate (10.73%). e compounds in leaf oil were also occupied by the same order, and the major compounds were 1,8-cineole (31.08%), α-terpineol (11.62%), and camphor (10%). e observations made in the current study gave a detailed profile of the essential oils from rhizome and leaf and also support the previous reports from different origins on the EO content of AC marked with 1,8cineole as the major component independent of the flowering or condition of the plants [39]. e most important chemical feature of the oils obtained from flowering stages of AC was the presence of a higher percentage of oxygenated monoterpenes, mainly 1,8-cineole and fenchyl acetate which were of industrial interest in terms of bioactivity.

Cytotoxicity and Proliferative Inhibition Effects of ACEOs.
Figures 2(a) and 2(b) show the effect of ACEOs on RAW 264.7, HaCaT, HepG2, and IEC-6 cell viability and proliferative ability. Treatment with 100 μg/mL caused a reduction in cell viability in all the above cell lines. erefore, ACEOs were considered at 50 μg/mL and below for further assays. Considering that intestinal epithelial cells and keratinocytes are the first point of contact when drugs are used orally or applied topically and virtually, all NSAIDs that have been used extensively are linked to at least rare cases of clinically apparent drug-induced liver injury [40]. Hence, the need to analyze the cytotoxicity becomes a necessity to identify safe and bioactive concentrations to mammalian cells for oral and topical curative applications. At 100 μg/mL, RAW 264.7, IEC-6, and HepG2 cells have less than 80% viability (Figure 2(a)). However, HaCaT cells were not affected at 100 μg/mL, which gives an interesting niche, suggesting a potential safe application of these ACEOs in topical therapeutics. Further, the proliferative inhibition efficacy of ACEOs was tested in order to explore the efficacy for prolonged inflammatory diseases, since chronic inflammation leads to neoplastic transformation of cells with the aid of proinflammatory markers [41]. Recent evidences on the TNF-α and IL-6-induced tumour progression of inflammatory cells [42] urge the search for anti-inflammatory drugs with tumour inhibition potential. In the present study, ACEOs exhibited inhibition of proliferation in a concentration-dependent manner in all the cell lines tested (Figure 2(b)). Among the 4 cell lines tested, HaCaT cells are the least affected with 23 ± 4.23 and 45 ± 3.45 percentage inhibition of proliferation at 50 μg/mL for RO and LO, respectively.

Inhibition of NO Release, Indirect iNOS Activation, and
Free Radical Nitrite Scavenging of ACEOs. ACEOs were tested for anti-inflammatory activity by the effect of them in Evidence-Based Complementary and Alternative Medicine LPS-induced NO production in murine macrophages. Figure 3 shows the concentration-dependent response of ACEOs towards the production of NO, indirect iNOS, and nitrite scavenging. LPS-induced RAW 264.7 cells produce large amounts of NO and the stable product, nitrite, which was measured by the colourimetric Griess assay. ACEOs exhibited significant inhibition of NO production at all concentrations tested in a concentration-dependent manner. EO from rhizome showed maximum inhibition of 85% at 50 μg/mL, whereas the leaf oil showed 81% inhibition. L-NMMA was used as a positive control at a concentration of 250 μM, and it exhibited NO inhibition of 87% when   Evidence-Based Complementary and Alternative Medicine RAW 264.7 cells were treated with LPS for 24 h. NO is a short-lived signaling molecule that plays an important role as an immunoregulatory mediator [43]. High NO levels cause a variety of pathophysiological processes including inflammation and carcinogenesis [44]. In addition, regulation of the iNOS-mediated release of NO from macrophages is considered as one of the strategies to develop therapeutics against various inflammatory diseases such as rheumatoid arthritis [45]. To investigate the involvement of iNOS in the mechanism of inhibition of NO production by ACEOs, RAW cells were treated with LPS for 12 h prior to the treatment with ACEOs, which showed a reduced NO production at the rate of 57.5% and 46.25% for rhizome and leaf, respectively, at 50 μg/mL. It also showed a   6). Symbols " * ," " * * ," and " * * * " represent a significant difference p < 0.05, p < 0.01, and p < 0.001, between LPS+ and EO-treated cells. Symbols " # " and " ## " represent a significant difference p < 0.05 between normal and LPS + cells, buffer, and SNP generated NO.   6). Symbols " * ," " * * ," and " * * * " represent a significant difference p < 0.05, p < 0.01, and p < 0.001, between LPS+ and EO-treated cells. Symbol " # " represents a significant difference p < 0.05 between normal and LPS + cells.

Evidence-Based Complementary and Alternative Medicine
concentration-dependent inhibition that further confirms the iNOS enzyme-mediated inhibition of NO production which facilitates the inhibition of iNOS gene expression at the genome transcriptional level. Further, ACEOs were tested for the scavenging activity of nitrite generated by SNP mediation that exhibited the moderate scavenging activity from rhizome (46.5%) and leaf (34.8%). is adds to the effect of ACEOs on NO inhibition which is by the inhibition of enzyme (iNOS) and not by mere scavenging activity.

Inhibition of Intracellular ROS Production and Superoxide
Scavenging Activity by ACEOs. ACEOs were studied for the inhibition of oxidative burst induced by opsonized zymosan   (OPZ) by a luminol-dependent chemiluminescence assay. Induction by OPZ caused the activation of NADPH oxidative complex which results in the production of ROS and this is mainly expressed in phagocytic cells of the immune system [46]. Prolonged local inflammatory reaction triggers the production of excessive amounts of ROS, and since macrophages have the highest burst capacity among the antigen-presenting cells, this harmful massive burst further triggers the immune activation of proinflammatory cytokine synthesis and results in tissue damage [47]. In the present study, the potent inhibitory activity was observed for the leaf EO (87% inhibition) and both the ACEOs exhibited a concentration-dependent inhibition of ROS production, as shown in Table 2. OPZ triggers both interferon-c and complement receptor activation producing superoxide anions that are spontaneously converted to halide ions by the oxidative enzymes [48] aggravating the oxidative burst. Luminol has its ability to enter the cell, which reacts with intracellular HOCl − exhibiting the ability of ACEOs in the inhibition of oxidative burst. In a separate experiment, superoxide anions were generated by incubating PMS with NADH, and the free radical O 2 − scavenging ability was studied in a quest to identify the extracellular superoxide scavenging activity of ACEOs. Interesting observation was obtained with the less inhibition pattern of 10-13% for the higher concentrations of ACEOs tested (Table 2). Earlier reports also indicated the essential oils as moderate scavengers of free radicals [49]. , and percentage inhibition of myeloperoxidase activity in tissue homogenates (c). N: normal; C: TPA-treated control group; RO: rhizome essential oil; LO: leaf essential oil; IMN: indomethacin at 0.5 mg/ear. Symbols " * ," " * * ," " * * * ," and " * * * * " represent a significant difference p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 between TPA treatment and EO-treated animals. Symbol " # " represents a significant difference p < 0.05 between normal and TPA-treated groups.

Inhibition of Cytokine Secretion by ACEOs.
recognizing the threat using the Toll-like receptor complex (TLR-4) which results in the intensified production of NO, ROS, and cytokines [50]. Intensified production of cytokines in the activated macrophages of the inflammatory process results in the tissue damage as seen in chronic inflammatory diseases [51]. Treatment with ACEOs significantly inhibited the production of cytokines by LPS (p < 0.05). Interestingly, ACEOs also decreased the proliferation of macrophages along with the inhibition of cytokine induction which makes it an effectual lead for the preparation of formulations for chronic disorders [52].

Effect of ACEOs on PGE2
Production. PGE2 is an eicosanoid lipid mediator produced when arachidonic acid is released from the plasma membrane by phospholipases and metabolized by two cyclooxygenases (COX 1 and COX 2) and three specific isomerases. NSAIDs act on COX enzymes, thus reducing the generation of PGE2. us, downstream pathways in the COX enzymes are responsible for response of PGE2 and are more specific targets in the treatment of inflammation and pain [53]. e effects of ACEO on the LPS-induced release of PGE2 from RAW 264.7 cells were studied. As PGE2 is one of the most important inflammatory mediators, the cells were pretreated with ACEOs for 2 h followed by incubation with 1 μg/mL of LPS. After 24 h of LPS treatment, the PGE2 contents in the culture medium were detected. e LPS-induced PGE2 secretion level was inhibited by treatment with the ACEOs at all the concentrations examined, and the maximum inhibition was observed at a concentration of 50 μg/mL for both oils ( Figure 5). Indomethacin (IMN) was used as a positive control. Diverse studies have proven that the expression of COX-2 is largely determined by transcriptional activation [54,55]. NF-κB, which is a mammalian transcription factor that regulates several genes and important in immunity and inflammation, can be triggered by LPS and other proinflammatory cytokines. In macrophages, NF-κB binds to COX-2 promoter and plays a role in LPS-mediated induction of COX-2. In addition, binding of CCAAT- enhancer-binding proteins (C/EBPs), c-AMP response element binding proteins (CREBs), and c-Jun to the COX-2 promoter ameliorates its transcriptional activation [56]. e present study is confined to the understanding of in vitro enzyme inhibitory activity of COX-1 and COX-2 and the production of PGE2; thence, it may be significant to understand the effect of ACEOs at the transcriptional activation level involving the NF-κB, C/EBP, CREB, and c-Jun proteins.

In Vitro COX Enzyme Inhibition by
ACEOs. COX-1 and COX-2 catalyze the biosynthesis of PGH2 from the AA substrate.
e COX-1 inhibition results in certain unsuitable side effects, whereas COX-2 inhibition provides therapeutic effects in pain, inflammation, cancer, Alzheimer's disease, and Parkinson disease [57]. erefore, the present study aimed at examining the COX-1 and COX-2 inhibitory activity of ACEO on purified enzymes as a mechanism of anti-inflammatory action. e oils showed inhibitory effects on COX-1 and COX-2 in a concentrationdependent manner (Tables 3 and 4). Furthermore, the concentrations that inhibited COX-2 had no effect on COX-1. e decrease in PGE2 production after ACEO treatment corresponded with the decrease in COX activity in vitro (COX-1 and COX-2), particularly COX-2. EOs of Illicium anisatum constituted mainly of 1,8-cineole and demonstrated its ability for inhibiting NO and PGE2 production in LPS-stimulated RAW 264.7 cells, along with the decrease in iNOS and COX-2 expression [58]. Further, Beer et al. [59] have studied the inhibitory effect of 1,8cineole on COX 1 and 2 activity and declared that 1,8 cineole is a potent and selective COX 2 blocker. Abundance of 1,8-cineole in ACEOs makes it an important candidate for its inhibitory activity.

Inhibition of Heat-Induced Hemolysis by ACEOs.
Reports on the mechanism of action of NSAIDs on antiinflammatory reaction are suggested to be exerted by stabilization of lysosomal membranes, which suppress the release of tissue-destroying enzymes which have been implemented in the pathogenesis of rheumatoid arthritis [60]. Protection against hypotonicity or heat-induced hemolysis by the red blood cell (RBC) membrane system is widely employed in the testing of drugs for anti-inflammatory activity [61]. Table 2 shows the percentage inhibition of RBC membrane lysis by the treatment of ACEOs. EO of leaf exhibited 83.32 ± 0.34% inhibition of RBC membrane lysis in closer rate to the dexamethasone (91.77 ± 0.56), commonly used drug in the autoimmune hemolytic anaemia [62] at 5 μM.

Effects of ACEOs on TPA-Induced Skin Inflammation.
Local application of TPA induces cutaneous inflammation and epidermal hyperplasia. Further, it stimulates infiltration of inflammatory cells, which releases large amounts of inflammatory mediators such as PGE2 and cytokines (TNF-α, IL-1β, and IL-6) [63]. In the current study, the therapeutic effect of ACEOs and its main constituents on TPA-induced skin inflammation was examined in a dose-dependent manner. Ear thickness was measured prior to the application of TPA. e concentrations of ACEO and compounds for testing were determined by doing a skin irritability test (data not shown). ACEO, CIN, and TPN presented significantly similar and effective anti-inflammatory activity in the experimental animal model used, which induced a strong dose-dependent edema inhibition (Figures 6(a) and 6(b) and Figures 7(a) and 7(b)). In particular, CIN showed a strong and similar edema inhibitory activity (29%) at 2.5% with indomethacin (25%) at 0.5 mg/ear. e mouse ear weight was reduced by 25% after indomethacin treatment, similar to rhizome EO (25%) and leaf EO (24.2%). TPN induced 23.4% edema reduction at 10% and 17% at 2% concentration. In summary, CIN and TPN which are the major compounds of AC presented a potent anti-inflammatory activity. To the best of our knowledge, it is the first report to demonstrate the topical anti-inflammatory activity of ACEO and also the compound TPN. Mounting evidences of the role of neutrophil enzyme, myeloperoxidase, in promoting oxidative stress in inflammatory pathologies and in many chronic inflammatory diseases such as atherosclerosis, glomerulonephritis, multiple sclerosis, rheumatoid arthritis, asthma, and cystic fibrosis makes it an important biochemical marker and a therapeutic target in antirheumatic drug development [64]. TPA-induced ear tissue homogenates showed high levels of MPO activity, and this activity was significantly reduced by the treatment with serial doses of ACEOs and compounds (Figures 6(c) and 7(c)). Further, the topical application also reduced the TNF-α, IL-1β, and IL-6 levels significantly in comparison with the TPA-induced group (Figures 8(a)-8(c)). In all the cases, rhizome oil exhibited more potential than the leaf oil (p < 0.05). Suggestive evidences on 1,8-cineole-and monoterpene-rich EOs on reducing airway inflammation, MPO activity, and decreased cytokine levels portrait the importance and efficacy of this topical study for therapeutic development [65]. Held et al. [28] have studied the oral anti-inflammatory effect of 1,8-cineole in various animal models, but the mechanism by which it exerts the activity was not clear. Our studies on the topical anti-inflammatory effect of CIN and TPN have explored the cytokine profile (Figures 9(a)-9(c)) and inhibition of MPO activity. From the results of this investigation, valuable research references can be provided for the clinical medicine or pharmaceutical application in the future.

Histopathological Analysis of Mouse Ear Tissue.
Further investigation on the H & E-stained ear biopsies from TPA-induced animals showed substantial increase in the ear thickness with vivid indication of edema, epidermal hyperplasia, and considerable neutrophil infiltration in the dermis associated with disruption in connective tissue. By comparison, 5% of ACEOs, CIN at 5%, and TPN at 10% treatment reduced ear thickness and affiliated pathological indicators to an extent comparable to the positive control, indomethacin, as shown in Figures 10 and 11. ese results directly exemplify the effects of ACEO and major compounds in the amelioration of TPA-induced contact dermatitis.

Effects of ACEOs on Formalin-Induced Pain and
Edema. Acute and chronic inflammatory diseases are associated with neuralgic as well as inflammatory pain [66].
Since topical drug formulations of NSAIDs are amongst the most frequently administered over-the-counter (OTC) drugs, the introduction of a new preparation of this group requires any comparison of its anti-inflammatory and analgesic efficacy to the corresponding standard [67]. 2.5% formalin injection to the mice paw shows a biphasic response and exhibits behavioral pain by flinching and licking of the affected paw [68]. e present study was carried out to test the analgesic and antiedema effect of ACEOs in a dose-dependent manner. Results of the present study indicate distinctly the topical analgesic effect of ACEOs by suppressing the pain induced by inflammation in the second   Data are mean ± SE. e numbers are frequencies (freq.) of flinching and total time (seconds; sec.) spent licking the formalin-injected paw. e behavioral response was measured at a total of 30 min after subplantar injection. p < 0.05 compared with the formalin-treated group (n � 6), # p < 0.05 compared with the normal group. Baseline values of paw volume represent preinjection diameters of paws. * p < 0.05, compared with the formalin-treated group (n � 6) (ANOVA with Tukey's test). RO: rhizome EO; LO: leaf EO. phase by significantly reducing the number of lickings and flinching ( Table 5). EO of leaf showed significantly greater activity (66%) in reducing the number of flinches and lickings compared to the rhizome oil (52%) (p < 0.05). Analgesic efficacy of aromatic plants and EOs is attributed to the major constituents (Table 6), and the synergism between such chemical constituents is always the main concern of pharmaceutical industries in the interest of pain management [69].

Conclusion
e composition of essential oils collected was rich in oxygenated monoterpenes and its major compounds were 1, 8cineole, α-Terpineol, and fenchyl acetate in rhizome, whereas leaf oil showcased the richness of camphor together with 1, 8-cineole and α-Terpineol. Further, ACEOs and main constituents ameliorate the production of inflammatory mediators such as nitric oxide, ROS, cytokines, and prostanoids in vitro and alleviate the edema and pain when applied topically in mice model of inflammation and pain. Further, ACEOs decrement the ontogeny of rat intestinal epithelial cells, human keratinocytes, and hepatocytes without affecting their viability. Hence, ACEOs featured the potential to be formulated into a more safe and sound alternative therapy for acute and chronic inflammatory maladies.