Intracellular ROS Scavenging and Anti-Inflammatory Activities of Oroxylum indicum Kurz (L.) Extract in LPS plus IFN-γ-Activated RAW264.7 Macrophages

Oroxylum indicum (L.) Kurz has been used as plant-based food and herbal medicine in many Asian countries. The aim of the present study was to examine the antioxidant and anti-inflammatory activities of O. indicum extract (O. indicum) in RAW264.7 cells activated by LPS plus IFN-γ. The phytochemical compounds in O. indicum were identified by GC-MS and LC-MS/MS. Five flavonoids (luteolin, apigenin, baicalein, oroxylin A, and quercetin) and 27 volatile compounds were found in O. indicum. O. indicum presented antioxidant activities, including reducing ability by FRAP assay and free radical scavenging activity by DPPH assay. Moreover, O. indicum also suppressed LPS plus IFN-γ-activated reactive oxygen species generation in RAW264.7 macrophages. It possessed the potent anti-inflammatory action through suppressing nitric oxide (NO) and IL-6 secretion, possibly due to its ability to scavenge intracellular ROS. The synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy results showed the alteration of signal intensity and integrated areas relating to lipid and protein of the activated RAW264.7 macrophages compared to unactivated cells. This is the first report of an application of the SR-FTIR technique to evaluate biomolecular changes in activated RAW264.7 cells. Our results indicate that O. indicum may be used as a potential source of nutraceutical for the development of health food supplement or a novel anti-inflammatory herbal medicine.


Introduction
Inflammation is a response of the immune system to injury, irritation, or infection caused by invading pathogens, radiation exposure, very high or low temperatures, or autoimmune processes. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, has been reported as one of the major causes of septic shock [1]. In response to endotoxin stimulation, a variety of immune cells can be activated. In the innate immunity system, macrophages play pivotal roles in the cellular host's defense against infection and tissue injury [2]. Inflammation is considered to be beneficial when it is short term and under control within the immune system (acute inflammation). However, if the inflammatory process has been going on for too long (chronic inflammation) or if the inflammatory response occurs in places where it is not needed, it can become problematic.
During inflammation responses, mast cells, monocytes, macrophages, lymphocytes, and other immune cells are first activated. e cells are recruited to the site of damage, resulting in the generation of reactive oxygen species that damage macromolecules, including DNA. At the same time, these inflammatory cells also produce large amounts of inflammatory mediators such as metabolites of arachidonic acid, nitric oxide (NO), proinflammatory cytokines, chemokines, prostaglandins, inducible enzymes, and growth factors [3]. Excessive production of intracellular ROS can cause oxidative stress associated with redox unbalance [4].
is imbalance leads to damage of essential biomolecules and cells, with potential impact on the whole organism. Accordingly, excessive oxidative stress and chronic inflammation can cause chronic diseases such as cancer, aging, diabetes, obesity, cardiovascular diseases, Alzheimer's, and Parkinson's disease. erefore, oxidative stress and inflammation must be adequately controlled to prevent the progressions of chronic diseases.
Over the past few decades, studies have investigated the possible protective role of plant foods against chronic diseases. Several epidemiological studies have revealed that higher consumption of fruits and vegetables is associated with a lower risk of chronic diseases [5]. e usage of herbs or traditional herbal medicines that are complementary and alternative medicines for the management of inflammation has increased because of the concerns about the adverse side effects of nonsteroidal anti-inflammatory drugs [6]. ese indigenous vegetables can be readily available in the local area. Consequently, the consumption of these vegetables would be appropriate to control and also reduce the cost of inflammatory management.
Oroxylum indicum (L.) Kurz is a species of flowering plant belonging to the Bignoniaceae family. Fruit pods of this plant have been used in many Asian countries as a plantbased food and herbal medicine for thousands of years without any known adverse effects [7][8][9]. Numerous scientific studies showed that O. indicum was not toxic even in experimental animals, even up to high doses [10][11][12]. e fruit pods of this plant are rich in nutrients concerning dietary fiber, essential amino acids, minerals, and fatty acids [8]. Every part of this tree is also an essential source of several medicinally essential flavonoids, including baicalein, chrysin, and oroxylin A, which have contributed to many of the biological activities. e crude extracts and its isolated compounds exhibit a broad spectrum of in vitro and in vivo pharmacological activities involving antioxidant, antidiabetic, anti-adipogenesis, hepatoprotective, and anti-inflammatory activities [7,11,13,14].
Currently, Fourier transform infrared (FTIR) microspectroscopy is widely used as a tool to monitor the changes in various biological samples such as bacteria, apoptotic and necrotic cell death, stem cell differentiation, and adipocyte cells [14][15][16][17]. FTIR technique is based on the absorption of infrared (IR) light of chemical bonds by vibrational transitions. is is a label-free and nondestructive technique that enables the analysis of biological samples with no need for staining and without sample destruction [18]. However, infrared microspectroscopy of cellular samples is not a single-molecule detection technique because of the limited spatial resolution when working on individual cells and the lack of sensitivity of the detectors while analyzing thick tissue sections [19]. In this context, the use of synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy was considered to explore the molecular chemistry within microstructures of individual single cells with a high signal [20]. To date, there are no studies available for SR-FTIR microspectroscopy to detect molecular changes in LPS plus IFN-c-activated RAW264.7 cells.
ere is considerable interest in applying SR-FTIR microspectroscopy to identify biochemical changes in LPS plus IFN-c-activated RAW264.7 cells. erefore, this study aimed to investigate the antioxidant and anti-inflammatory activities of O. indicum in RAW264.7 cells activated by LPS plus IFN-c. We also examined the biochemical alteration in LPS plus IFN-c-activated RAW264.7 cells upon treatment with O. indicum by using SR-FTIR microspectroscopy.

Preparation of Plant Extract.
e fresh fruit pods of O. indicum were purchased from the local market at Wang Nam Khiao District, Nakhon Ratchasima province, ailand. e plant samples were identified by a botanist at the Institute of Science, Suranaree University, ailand, and the voucher specimens were kept at the flora of Suranaree University of Technology Herbarium (SOI0808U). Fresh fruit pods were washed and cut into small pieces and then dried in the oven at 40°C for 2 days. e dried pieces were pulverized using a mechanical grinder. O. indicum dry powder (500 g) was extracted with 95% ethanol by a soxhlation for 8 h and then filtered through Whatman filter paper. e ethanolic extract was concentrated and lyophilized to obtain the powder of O. indicum. e crude extracts were stored at −20°C till use in subsequent experiments. O. indicum was dissolved in 100% DMSO and diluted to 0.06% (v/v) in the cell culture medium.

GC-MS Analysis.
e phytoconstituents present in the extract of O. indicum were analyzed by gas chromatographymass spectrometry (GC-MS) using a Bruker 450-GC/Bruker 320-MS equipped with Rtx-5MS fused silica capillary column (30 m × 0.25 mm; 0.25 μm in the thickness of film). Helium was used as the carrier gas with a flow rate of 1 mL min −1 . e injector temperature was operated at 250°C, and the oven temperature was programmed from 110°C (2 min), 200°C (3 min), and 280°C (20 min) with rates of 0, 10, and 5°C min −1 , respectively. e mass spectral data were taken with an electron energy of 70 eV. e ion source and transfer line temperature were kept at 200°C. e mass spectra were obtained by a centroid scan of the mass range from 45 to 500 atomic mass units. Interpretation of the mass spectrum of GC-MS was made by using NIST mass spectral library 2008.

Liquid Chromatography-Mass Spectrometer (LC-MS/MS) Quantification of the Selected Phenols and Flavonoids.
e analyses were performed on the Dionex Ultimate 3000 UHPLC system (Dionex, USA) coupled with electrospray ionization (ESI) tandem mass spectrometer (micro-TOF-Q II) (Bruker, Germany). e injection volume of all samples was 5 μL. e separation was achieved using a Zorbax SB-C18 (250 mm × 4.6 mm × 3.5 μm (Agilent Technologies, USA)) and thermostat at 35°C, with a flow rate of 0.8 mL min −1 of the mobile phase, which included deionized water containing 0.1% formic acid (FA) as solvent A and acetonitrile containing 0.1% formic acid as solvent B. e gradient elution was performed using the following solvent gradient: starting with 30% B, reaching 80% B at 30 min, and holding until 38 min, reducing to 30% B in 2 min and holding until the run ending at 45 min. Detection of eluted components, ionized by ESI, was performed in the mass scanning mode in the range of 50 m/z to 1,500 m/z at negative ion polarity. e nebulizer gas (N 2 ) was 2 Bar, drying gas was 8 L min −1 , the dryer heater temperature was 180°C, and the capillary voltage was 4.5 kV. e LC-QTOF data were collected and processed by Compass 1.3 software (Bruker, Germany). e target phenolic and flavonoid compounds were identified and quantified with Bruker Quant Analysis Version 2.0 SP 5 software. e calibration curves were constructed from peak areas of different concentrations of the reference standard (from 0.5 μg mL −1 to 250 μg mL −1 ), and the concentration of targeted compounds was calculated based on the equation for linear regression obtained from the calibration curves.

Ferric-Reducing/Antioxidant Power (FRAP) Assay.
FRAP is based on the detection of the sample capacity to reduce ferric ions, which is measured as a change in the absorbance of the ferrous TPTZ complex.
e assay was carried out, according to Rupasinghe et al. [21]. Briefly, the working FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), a solution of 10 mM 2,4,6-tripyridyls-triazine (TPTZ) in 10 mM hydrochloric acid and 20 mM ferric chloride at 10 : 1:1 (v/v/v). e working FRAP reagent (180 μL) and sample solutions (20 μL) were mixed in a 96well plate for 6 min. e absorbance was measured at 595 nm by using a microplate reader (Bio-Rad Laboratories, Inc., USA). e standard calibration curve was developed using different concentrations of Trolox and VIT.C. FRAP values were expressed as a milligram of Trolox equivalent antioxidant capacity (TREA) or ascorbic equivalent antioxidant capacity (VCEA) per gram of dry extract.

DPPH Radical Scavenging Activity Assay.
e total free radical scavenging capacity of O. indicum was estimated according to the method of Yang et al. [22]. Briefly, one hundred microliters of the sample at different concentrations were added to 100 μL of DPPH solution (0.2 mM) in a 96-well plate. e mixture was shaken vigorously at room temperature for 15 min in the dark and measured the absorbance at 517 nm by using a microplate reader (Bio-Rad Laboratories, Inc, USA). Trolox and VIT.C were used as a positive control. e free radical scavenging activity was calculated as follows: e IC 50 of DPPH_was determined from a dose-response curve using linear regression analysis. Decreasing DPPH solution absorption indicates an increase of DPPH radical scavenging activity.

In Vitro Cytotoxic Test (MTT Assay).
e cytotoxic effect of O. indicum on cell viability was determined by using a tetrazolium dye (MTT) colorimetric assay [23]. Briefly, the cells were seeded in a 96-well plate at a density of 2 × 10 4 cells/well and allowed to adhere for 24 h. Cells were treated with different concentrations of O. indicum for 24 h. After incubation, the culture medium was removed, and 0.5 mg mL −1 (final concentration) MTT was added. en, the cells were further incubated for 4 h at 37°C. Formazan crystal formed by viable cells was dissolved in DMSO and absorbance was measured at 540 nm with a microplate spectrophotometer (Bio-Rad Laboratories, Inc., USA).

Assessment of Intracellular ROS Scavenging Activity.
e intracellular ROS scavenging capacity of O. indicum in RAW264.7 cells induced by LPS plus IFN-c was measured using a DCFH-DA fluorescent probe, according to the Evidence-Based Complementary and Alternative Medicine method described by Sittisart and Chitsomboon [23]. Briefly, RAW264.7 cells were seeded in a 96-well black plate at 2.0 × 10 4 cells/well and incubated overnight. en, the culture medium was removed, and the cells were pretreated with O. indicum at the concentration of 50, 100, or 200 μg mL −1 , VIT.C 50 μg mL −1 , baicalein 5 μg mL −1 , or a selective ROS scavenger, NAC, for 3 h. en, the cells were activated with 1 μg mL −1 LPS plus 10 ng mL −1 IFN-c and another 24-hour incubation. After removing the medium, the cells were treated with 20 M DCFH-DA in Hank's Balanced Salt Solution (HBSS) for 30 min and then washed with PBS twice. e fluorescence intensity was measured using a Gemini EM fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. e percentage of DCF fluorescence intensity was calculated by the following formula: DCF fluorescence intensity (%) � (DCF fluorescence intensity test group /DCF fluorescence intensity control group ) × 100. e IC 35, IC 40 , and IC 50 of O. indicum were also calculated from a dose-response curve using linear regression analysis.

Determination of Nitric Oxide (NO) and Proinflammatory Cytokines (IL-6 and TNF-α) Production.
e anti-inflammatory activities of O. indicum were evaluated by measuring the level of NO and proinflammatory cytokines (IL-6 and TNF-α) production in LPS plus IFN-c-activated RAW264.7 cells. e cells were seeded at a density of 6 × 10 5 cells/well in a 6-well plate and then incubated overnight. After incubation, the culture medium was removed, and the cells were pretreated with different concentrations of O. indicum (50, 100, and 200 μg mL −1 ) or the anti-inflammatory agent, DEX (1 μM), for 3 h. en, the cells were activated with 1 μg mL −1 LPS plus 10 ng mL −1 IFN-c and incubated for 24 h. e supernatant was collected for further analysis of NO using Griess reagent and determined the level of TNF-α and IL-6 with the ELISA kits. e level of NO in the culture media was detected as nitrite, a major stable product of NO, using Griess reagent as described by Sittisart et al. [24]. Briefly, 100 μL of cell culture medium was mixed with an equal volume of Griess reagent in a 96-well plate and incubated at room temperature for 10 min in the dark. e intensity of the pink color of the samples was measured at 540 nm using a microplate reader (Bio-Rad Laboratories, Inc.). e amount of nitrite in the samples was determined using the linear sodium nitrite calibration curves at a concentration range of 2.5-100 μM.
Proinflammatory cytokine levels (IL-6 and TNF-α) were quantified by Mouse IL-6 or Mouse TNF-α DuoSet ® ELISA Kits (R&D systems Inc., Minneapolis, USA) according to the manufacturer's instructions. Optical density was measured at 450 nm with a microplate reader (Benchmark Plus, Bio-Rad, Japan). By preparing the standards of known cytokine concentrations, the number of cytokines in the samples was quantified from a standard curve.

Haematoxylin
Staining. e macrophage cells were activated by LPS plus IFN-c for 24 h and then stained with a haematoxylin solution to observe the phenotype feature as described by Dunkhunthod et al. [13] with some modification. Shortly, the cells were washed with PBS twice and fixed with 10% formaldehyde in PBS for 1 h. en, cells were washed with distilled water twice and stained with haematoxylin solution for 10 min at room temperature. e stained cells were visualized under the inverted fluorescence microscope (Olympus Corporation, Japan).

FTIR Measurement Using Synchrotron IR Source.
e sample was collected and dropped onto a barium fluoride (BaF 2 ) optical window (Crystran, Crystran Ltd.) as previously described by Dunkhunthod et al. [13]. FTIR experiments were conducted using a spectroscopy facility at the Synchrotron Light Research Institute (Public Organization), ailand. FTIR spectra were acquired in transmission mode with a Vertex 70 FTIR spectrometer coupled with a Bruker Hyperion 2000 microscope (Bruker Optics Inc., Ettlingen, Germany), using synchrotron radiation as an IR source. e microscope was equipped with 64×64 element MCT, FPA detector, which allowed simultaneous spectral data acquisition with a 36 × objective. FTIR spectrum was recorded within a spectral range of 4000-600 cm −1 using an aperture size of 10 μm × 10 μm with a spectral resolution 4 cm −1 , with 64 scans being coadded. OPUS software (Bruker Optics Ltd., Ettlingen, Germany) was used for spectral measurement and instrument control. e preprocessing of the spectra was performed by second derivative transformations using the Savitzky-Golay algorithm (nine smoothing points) and normalized with extended multiplicative signal correction (EMSC) using the spectral regions from 3000 to 2800 cm −1 and 1800 to 1400 cm −1 . A principal component analysis (PCA) was performed using the Unscrambler ® 10.5 software packages (CAMO Software AS., Oslo, Norway). Score plots (3D) and loading plots were used to represent the different classes of data and relations among variables of the data set, respectively. e integrated peak areas of FTIR spectra were analyzed using OPUS 7.2 software (Bruker) in the lipid regions (3000-2800 cm −1 ) and protein regions (1800-1400 cm −1 ) and demonstrated on a histogram.

Statistical Analysis.
All data are expressed as the means ± SD from at least three independent experiments. e statistical significance (Statistical Package for the Social Sciences, version 19) was determined by performing a oneway analysis of variance (ANOVA) with Tukey's post hoc analysis to determine the differences among each treated group. Statistical significance was considered at p < 0.05.  Table 1.

Free Radical Scavenging and Antioxidant Activities of O. indicum.
e antioxidative potential of O. indicum was assessed in vitro by DPPH and FRAP assays. e obtained results are shown in Table 2. In this study, the free radical scavenging activity of O. indicum and standard compound, VIT.C and Trolox, were determined using the DPPH-based method.
e results showed that the DPPH scavenging ability of O. indicum and standard compound, VIT.C, were comparable and both significantly stronger than Trolox (p < 0.05). e ferric reducing antioxidant power (FRAP) was used to assess whether O. indicum had an electrondonating capacity. O. indicum exhibited a degree of electrondonating capacity by 57.14 ± 4.39 μgVCEA mg −1 and 65.77 ± 4.99 μgTREA mg −1 of dry extract. ese results indicate that O. indicum displays an antioxidant activity based on the reducing ability to reduce ferric ion (Fe 3+ ) to ferrous ion (Fe 2+ ). erefore, it could be concluded that O. indicum displays strong antioxidant activity in the assay used in this

Effects of O. indicum on Intracellular ROS Production in
LPS plus IFN-c-Activated RAW264.7 Cells. In the event of the inflammatory response, the classically activated macrophages respond to intracellular pathogens by secreting proinflammatory cytokines, chemokines, proteases, and the production of reactive oxygen species [29]. ese factors are key signaling molecules that play a significant role in host defense and inflammation. e overproduction of ROS can prompt injury issues that might initiate the inflammation process [30]. Ribeiro et al. reported that flavonoid compounds possessed anti-inflammatory activity by the modulation of ROS generated through the neutrophils' oxidative burst [31]. erefore, a flavonoid-enriched extract from O. indicum undoubtedly contributes to their anti-inflammatory roles by Values are mean ± SD (n � 3) and are representative of three independent experiments with similar results. Different letters within the same column are significantly different at p < 0.05.  scavenging intracellular ROS and thus be useful for preventing the uncontrolled inflammation process. To investigate whether the protective effects of O. indicum on the LPS plus IFN-c-induced inflammatory response were due to a blockade of oxidative stress, the intracellular ROS scavenging potential of O. indicum was evaluated in LPS plus IFN-c-activated RAW264.7 cells. As presented in Figure 3, the treatment of RAW264. 7  Previous investigators demonstrated that baicalein enhanced cellular antioxidant defense capacity in C6 glial cells by inhibiting ROS production and activating the Nrf2 signaling pathway [34]. Qi et al. [35] found that baicalein reduced LPS-induced inflammation via suppressing JAK/ STATs activation and ROS production. In addition, baicalein reduced H 2 O 2 -induced DNA damage as a result of a decrease in phospho-H2A.X production, DNA tail formation, and lipid peroxidation prevention [36]. Our results indicated that the pretreatment of RAW264.7 cells with O. indicum at a concentration of 200 μg mL −1 significantly decreased the intracellular ROS accumulation by approximately 64%. is finding indicates that O. indicum at 200 μg mL −1 , which contains baicalein around 5 μg mL −1 , shows significantly stronger radical scavenging potency than baicalein alone (p < 0.05, Figure 3). e higher potency of O. indicum may be due to the other bioactive compounds present in O. indicum such as c-sitosterol, stigmasterol, luteolin, apigenin, and quercetin, which could act as ROS scavenging agents. All compounds above have been shown to possess antioxidant properties. e intracellular ROS scavenging potency of O. indicum is more than two times stronger compared to baicalein alone which leads us to believe that synergistic activity could take place between baicalein and other flavonoids or volatile compounds [37].

Effects of O. indicum on Nitric Oxide (NO) and Proinflammatory Cytokine (IL-6 and TNF-α) Production in LPS
plus IFN-c-Activated RAW264.7 Cells. During inflammation, the macrophages actively participate in the inflammatory response by releasing cytokines (TNF-α, IL-1β, and IL-6), chemokines, and inflammatory mediators (NO, iNOS, PGE 2 , and COX-2) [38]. e overproduction of these agents contributes to the induction and progression of several inflammatory diseases.
us, it is crucial to regulate the inflammatory mediators in controlling the inflammatory progression and treating inflammatory disorders. Nitric oxide (NO) is synthesized by many cell types involved in immunity and inflammation. NO is vital as a toxic defense molecule against infectious organisms. On the other hand, NO reacts rapidly with superoxide to form the more reactive product, peroxynitrite (ONOO − ), which can directly react with various biological targets and components of the cell, including lipids, thiols, amino acid residues, DNA bases, and low-molecular-weight antioxidants [39]. erefore, the anti-inflammatory potential of O. indicum on inhibition of NO production was measured after the treatment of O. indicum in LPS plus IFN-c-activated RAW264.7 cells. e anti-inflammatory agent, DEX, was selected to serve as the reference drug. As shown in Figure 4(a), upon LPS plus IFN-c treatment (CON), NO production was increased with the nitrite level peaking to 54.17 ± 0.38 μM. However, pretreatment of cells with the highest concentration (200 μg mL −1 ) of O. indicum suppressed the production of NO of about 16%, which is precisely the same efficiency as 1 μM DEX, compared to the LPS plus IFN-c-activated group.
Qi et al. reported that baicalein suppressed LPS-induced inflammatory responses in RAW264.7 macrophages via attenuating NO synthesis [35]. Shimizu et al. noted that an equimolar mixture (F-mix) of baicalein, wogonin, and oroxylin A showed a synergistic inhibitory effect on the production of NO in LPS-treated J774.1 cells [40]. Also, a mixture of β-sitosterol and stigmasterol isolated from Andrographis paniculata significantly suppressed NO production in LPS/IFN-c stimulated RAW264.7 cells [41].
Both IL-6 and TNF-α are essential proinflammatory cytokines, either of which can serve as an indicator of inflammation. To confirm the anti-inflammatory effect of O. indicum, we also investigated the inhibitory effect of O. indicum on proinflammatory cytokine secretion by measuring IL-6 and TNF-α levels in RAW264.7 cells activated by LPS plus TNF-α. Exposure of the cells with LPS plus IFN-c strongly activated the secretion of IL-6 and TNF-α compared to the unactivated group (Figures 4(b) and 4(c)) [42]. e results showed that DEX markedly reduced the secretion of IL-6 and TNF-α of about 69.34% and 62.02%, respectively, compared to the LPS plus IFN-c-activated group. Treatment with O. indicum inhibited the secretion of IL-6 in a dosedependent manner (Figure 4(b)), but it did not affect the reduction of TNF-α level (p > 0.05, Figure 4(c)). e concentration of O. indicum at 200 μg mL −1 exerted an IL-6 inhibition by 62.99%, which was practically similar to the reference drug, DEX (69.34% inhibition).
Other researchers had demonstrated that the ethyl acetate extract derived from the stem bark of O. indicum showed the inhibitory effect on LPS-induced IL-6, IL-1β, and TNF-α release in human monocytes [7]. In vivo study indicated that the aqueous decoction of both stem bark and root bark of O. indicum produced anti-inflammatory activity by reducing paw edema formation in the carrageenan-induced paw edema model [42]. Furthermore, flavonoids found in this plant, such as baicalein, apigenin, oroxylin A, and luteolin, are known for their anti-inflammatory effects attributed at least partially through the suppression of proinflammatory cytokines, IL-6, IL-1β, and TNF-α [43].
Moreover, it had been reported that a mixture of β-sitosterol and stigmasterol isolated from Andrographis paniculata significantly decreased TNF-α and IL-6 secretion from LPS/ IFN-c-stimulated RAW264.7 cells [41]. ese findings provide evidence that the main constituents of O. indicum are flavonoids and phytosterols, which could potentially act as the anti-inflammatory compounds of this plant.
Differential effects of O. indicum on IL-6 and TNF-α production in RAW264.7 cells could be explained by the different mechanisms of O. indicum on the secretion of IL-6 and those of TNF-α alleviation. Several studies reported that the mechanism, signaling pathways, and transcription factors controlling TNF-α expression were distinct from those of IL-6 [44][45][46]. It had been reported that the activation of p38 MAPK was required for the LPS/TLR4-induced expression of TNF-α, but not IL-6 [44]. It is well known that CREB recognizes a similar DNA binding sequence in the promoter region of the TNF-α gene [47]. In contrast, similar sequences have not been identified on the IL-6 and IL-1 promoters. Also, the STAT3 tyrosine phosphorylation is a key role to induce IL-6 production in response to inflammation. In vitro study revealed that the blocking STAT3 activity preferred to inhibit LPS-mediated production of IL-1β and IL-6, but not TNF-α, in RAW264.7 cells [46]. e study of Prêle et al. also confirmed that the STAT3 activation did not directly regulate LPS-induced TNF-α production in human monocytes [48].  (Figure 5). e unactivated RAW264.7 cells showed the round morphology, whereas LPS plus IFN-c-activated RAW264.7 cells showed enlargement, dendritic, spindle, and spheres, which were the phenotype features of activated macrophages [49]. e previous study indicated that the morphological changes of adherent macrophages were associated with their secretion of inflammatory cytokines [50]. ese cells treated with O. indicum at 200 μg mL −1 displayed less activated macrophage phenotypes than activated RAW264.7 cells, DEX-and O. indicum treated at 50 and 100 μg mL −1 .  Evidence-Based Complementary and Alternative Medicine e raw spectrum was more useful to perform the second derivative analysis in spectral regions ranging from 3,000 to 2800 cm −1 for lipid regions and from 1800 to 1400 cm −1 for protein regions, as shown in Figure 6(b). e signal intensity and area of the peaks of protein and lipid regions of these groups are calculated and shown in Figure 6(c). Moreover, the FTIR band assignments are shown in Table 3. e change in cellular lipid was observed in the mainly lipid region (3000-2800 cm −1 ). e average second derivative spectra of RAW264.7 cells under different experimental conditions exhibited three specific regions at 2960 cm −1 , 2921 cm −1 , and 2850 cm −1 , which are assigned to the asymmetrical stretching vibrations of the CH 3 and CH 2 groups of the phospholipids membrane and CH 2 symmetric stretching, respectively (Figure 6(b)). e relative absorbance at 2960 cm −1 , 2921 cm −1 , and 2850 cm −1 in activated RAW264.7 cells, DEX-, or O. indicum-treated activated RAW264.7 cells was higher than that in the unactivated RAW264.7 cells. e integrated areas of lipid regions of the second derivative spectra were calculated for unactivated and treated activated RAW264.7 cells in order to quantify the lipid content [49]. e results exhibited that the integrated area of the lipid region was significantly increased in activated RAW264.7 cells, DEX-, and O. indicum-treated activated RAW264.7 cells compared to the unactivated RAW264.7 cells (p < 0.05). e increase of lipid regions in LPS plus IFN-c-activated RAW264.7 cells under three different experimental conditions could be related to cell membrane changes. e changes in the membrane are due to phenotype features of activated macrophage, including enlargement, dendritic, and spherical features ( Figure 5), which developed in response to the inflammatory inducer during inflammation [49]. Moreover, these results are consistent with Funk et al.'s study [51] showing that the activation of RAW264.7 macrophages enhances their ability to accumulate lipid from a variety of lipid molecules and to become foam cells. e cellular protein change was observed within 1800 cm −1 and 1400 cm −1 interval, reflecting the vibrations of the amide I (1700-1600 cm −1 ) and amide II (1600-1400 cm −1 ) (Figure 6(b)). In unactivated RAW264.7 cells, a strong peak at 1655 cm −1 and 1545 cm −1 assigned to stretching vibrations of α-helix secondary protein structure displayed higher signal intensity than other groups. ese results are in accordance with the integrated areas of the protein regions where the protein content in unactivated RAW264.7 cells is significantly greater than that in the activated RAW264.7 cells and DEX-or O. indicum-treated activated RAW264.7cells (p < 0.05, Figure 6(c)). us, these results reflect the altered cellular protein profile in all treated groups of LPS plus IFN-c-activated RAW264.7 cells compared to unactivated RAW264.7 cells. Upon treatment of cells with O. indicum, the protein content was significantly increased compared to LPS plus IFN-c-activated RAW264.7   inhibition [52]. Accordingly, the oxidized proteins thus become better targets for proteolytic digestion by the 20S proteasomes and, consequently, decrease the levels of the proteins in general [53]. Flavonoids play an important role as a ROS scavenger by locating and neutralizing radicals before they damage the cell structure [54]. erefore, the flavonoid-enriched extract from O. indicum could protect proteins from oxidation under oxidative stress, and proteolytic digestion leads to an increase in cellular protein contents.

Effects of O. indicum on Biomolecular Changing
However, there was no significant difference between the FTIR spectra of unactivated RAW264.7 cells and other groups in nucleic acid regions (1,300-900 cm -1 ) (data are not shown). e data indicated that the selected concentration of O. indicum did not produce any effect on nucleic acids (DNA and RNA base) of the cells, which was consistent with PCA score plots showed distinct clustering between unactivated RAW264.7 cells (n � 120), activated RAW264.7 (LPS plus IFN-c) (n � 120), and activated RAW264.7 (LPS plus IFN-c) exposed to 1 μM DEX (n � 157) or 200 μg mL −1 of O. indicum (n � 135). PCA loading plots identify biomarker differences over a spectral range of samples.
its cytotoxicity evaluation by using MTT assay. Supporting our results, Zelig et al. reported that a decrease in DNA absorbance was associated with apoptotic cell death, by contrast, to increase during necrotic cell death [55]. In order to discriminate the distinct clustering of spectra from the four cell populations, PCA analysis was performed to identify which wavenumbers in the FTIR spectra complex showed the largest spectral variation within the sample. e 3-dimensional PCA clustering results from FTIR spectral data of unactivated RAW264.7 cells, activated RAW264.7 cells, and activated RAW264.7 cells after treatment with DEX or O. indicum are displayed in Figure 7(a). e PCA score plot demonstrated that the clusters of unactivated RAW264.7 cells and O. indicum-treated activated RAW264.7 cells were separated from activated RAW264.7 cells and DEX-treated activated RAW264.7 cells along PC1 (54%) and PC4 (6%) whereas the clusters of unactivated RAW264.7 cells were distinguished from O. indicum-treated activated RAW264.7 cells along PC2 (10%). e PCA loading plots (Figure 7(b)) were used to identify the regions of the spectrum, which most contributed to the clustering. e positive score of the spectra of the unactivated RAW264.7 cells and O. indicum-treated activated RAW264.7 cells was clearly separated from the negative score of the spectra of the other two groups along with PC1 score plot, which displayed remarkably high negative PC1 loadings at 1660 cm −1 and 1552 cm −1 (suggesting an α-helix protein structure of amide I and amide II, respectively). PC2 loading plot was discriminated by the negative loading spectra at 2919 cm −1 and 2852 cm −1 caused by the C-H stretching assigned to the lipids and at 1693 cm −1 and 1668 cm −1 resulting from the α-helix protein structure of amide I, which separated the positive score of DEX-treated activated RAW264.7 cells from the negative score of the unactivated RAW264.7 cells. e amide I band from proteins at 1642 cm −1 (assigned to the α-helix structure) and the C-H stretching region (assigned to the lipids) were heavily loaded for PC4 which separated the positive score of the spectra of the unactivated RAW264.7 cells and O. indicumtreated activated RAW264.7 cells from the negative score of the spectra of the activated RAW264.7 cells and DEX-treated activated RAW264.7 cells. ese results are consistent with its second derivative spectra and the integrated areas of protein and lipid regions.

Conclusions
In conclusion, these findings provide evidence that O. indicum could possess the antioxidant and anti-inflammatory effects in LPS plus IFN-c-activated RAW264.7 cells by scavenging intracellular ROS, reducing NO and IL-6 secretion, respectively. ese pharmacological properties of O. indicum may occur from the synergistic interaction between its flavonoids and phytosterol components. ese results also provide the first evidence of the potential use of SR-FTIR microspectroscopy to evaluate the biochemical profile alteration of activated RAW264.7 macrophages. erefore, O. indicum may be used as a potential source of nutraceutical for the development of health food supplement or a novel anti-inflammatory herbal medicine to alleviate the excessive inflammatory response.
Data Availability e datasets used and analyzed during this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this manuscript.