2021 Volume 27 Issue 1 Pages 111-119
Dragon fruit is a prebiotic source of oligosaccharides. This is the first report on a study that evaluates the anti-inflammatory effects of dragon fruit oligosaccharides (DFO) on a macrophage cell line. This experiment evaluated the effects of DFO on both gene and protein expression levels in the inflammatory response of a lipopolysaccharide-induced RAW 264.7 macrophage cell line. The results showed that DFO significantly inhibited prostaglandin-E2, nitric oxide, and tumor necrosis factor-α production. DFO also significantly suppressed the expression of cyclooxygenase-2 and inducible nitric oxide synthase. These results suggest that DFO is a potentially novel ingredient for use as an anti-inflammatory prebiotic for the prevention of inflammation.
Inflammation is an energetic tissue defense response to damaging stimuli, and an attempt by the organism to rid itself of the stimulus and then initiate the tissue healing process (Wang et al., 2020). Lipopolysaccharides, which are endotoxins derived from the outer membrane of Gram-negative bacteria, can recruit a group of inflammatory mediators, among which are macrophages, to inflammatory sites and initiate intracellular cascades (He et al., 2018). Macrophages perform a critical role in the immune response of organisms via the excretion of a series of pro-inflammatory cytokines, Nitric Oxide (NO), signaling proteins and several other inflammatory mediators) and are activated by stimuli, such as pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) (Muniandy et al., 2018). NO is regulated by inducible NO synthase (iNOS) and reacts with peroxides to boost inflammatory processes (Kwon et al., 2018). In addition, cyclooxygenase-2 (COX-2) is a key inflammatory enzyme that is involved in forming prostaglandin-E2 (PGE2) from arachidonic acid (Park et al., 2018). However, these inflammatory mediators are only expressed in low quantities in most normal tissues, but their expression is powerfully up regulated by LPS and other stimuli (Li et al., 2018).
Prebiotics are non-digestible carbohydrates, dietary fibers, and oligosaccharides, which serve as a source of food for bacteria in the gut system. (Gibson et al., 2017). Previous studies have shown that prebiotics, such as fructo-oligosacharide and inulin (Vogt et al., 2013), mannan oligosaccharide (MOS) (Ferenczi et al., 2016), neoagaro-oligosaccharides (NAOS) (Wang et al., 2017), and pectin oligosaccharide (POS) (Tan et al., 2018), are capable of directly modulating the immune system including anti-inflammatory responses. The direct pathway involves the activation or inhibition of cellular receptors on inflammatory cells, such as toll-like receptor 4 (TLR4) and other TLRs, e.g., 2, 3 and 5 via the nuclear factor kappa B (NF-κB) pathway (Tan et al., 2018). Our previous reports based on in vitro models revealed that dragon fruit contains oligosaccharides with prebiotic properties (Wichienchot et al., 2010; Dasaesamoh et al., 2016; Pansai et al., 2020). Moreover, it has been reported that in in vivo studies, DFO exerted immune-boosting properties in normal rats (Pansai et al., 2020).
To the best of our knowledge, there have been no previous reports on the effects of DFO on the inflammatory response in cell culture studies. Thus, this study reports the inflammatory response observed in terms of PGE2, NO and TNF-α production and also gene expression of enzymes including COX-2, iNOS and TNF-α, in LPS-induced inflammation after pre-treatment with DFO in RAW 264.7 cells.
DFO preparation Dragon fruit oligosaccharides (DFO) was derived from dragon fruit (Hylocereus undatus (Haw.) Britt. & Rose). The chemical composition, oligosaccharide contents and prebiotic properties of DFO powder was confirmed using gut model as described by Pansai et al. (2020). The carbohydrate content consists mainly of oligosaccharides and fructose. The oligosaccharide contents analyzed by HPAEC-PAD, revealed that the DFO powder consists of several DP including DP3 (raffinose), DP4 (stachyose) and DP5 (maltopentaose). These oligosaccharides were predominantly DPs of 3 to 5, with fructan-type composition such as fructo-oligosaccharides (FOS). The commercial FOS was selected as a comparable prebiotic in order to evaluate inflammatory responses on RAW 264.7 cells. The commercial FOS has total FOS > 95% (HONNE CO., LTD., China). The dragon fruit oligosaccharides and commercial FOS powder were dissolved in DNase-RNAse free water, filtered through 0.22 µm of membrane filter, and prepared at a stock concentration of 100 mg/mL.
Cell culture The RAW 264.7 macrophage cell line was purchased from American Type Culture Collection (ATCC, USA). The cells were cultured in RPMI-1640 medium containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) (Invitrogen, USA). All cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2.
Cytotoxicity assay The cells were plated overnight in 96-well plates (5 × 104 cells/well), with different concentrations of DFO extract being added to the wells. After 24 h, 10 µL of 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide solution (5 mg/mL) (Invitrogen, USA) was added, followed by 2 h incubation at 37 °C. Then, the culture supernatant was discarded and 100 µL Dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader.
Nitric oxide (NO) determination RAW 264.7 cells were pre-incubated with DFO at various concentrations or fructo-oligosaccharides (FOS) in 6-well plates (5 × 105 cells/well) for 24 h. This was followed by incubating either with or without Lipopolysaccharides (LPS) (1 µg/mL) (Sigma-Aldrich, USA), for another 24 h. The nitrite accumulation in the culture medium was measured using the Griess method (Merck, Germany). Briefly, 100 µL of cell-culture supernatant was mixed with 100 µL of Griess reagent in a 96-well plate, incubated at room temperature for 10 min, and then measured at 540 nm using a microplate reader.
Measurement of TNF-α and PGE2 production by ELISA assay RAW 264.7 macrophage cells together with DFO at various concentrations or FOS were seeded in 6-well plates (5 × 105 cells/well) for 24 h. After that, LPS (1 µg/mL) was added and the cultures were incubated for 24 h. The levels of TNF-α and PGE2 accumulation in the culture medium were determined with specific ELISA kits according to the manufacturer's instructions (PeproTech, USA and R&D systems, USA, respectively).
Measurement of the mRNA expression of COX-2, iNOS and TNF-α by qPCR Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA was reversely-transcribed into cDNA with an iScriptTM Reverse Transcription Supermix for cDNA Synthesis Kit (Bio-Rad, Singapore) and quantification of TNF-α, iNOS, and COX-2 expression was performed with SsoFastTM EvaGreen®Supermix real time PCR MasterMix (Bio-Rad, Singapore) and the data was presented as 2−ΔΔct. GAPDH was used as an internal control and the primers used are shown in Table 1.
| Gene | Name | Primers sequence |
|---|---|---|
| COX-2 | Forward Primer | 5′-GGAGAGACTATCAAGATAGTGATC-3′ |
| Reverse Primer | 5′-ATGGTCAGTAGACTTTTACAGCTC-3′ | |
| iNOS | Forward Primer | 5′-ATCTGGATCAGGAACCTGAA-3′ |
| Reverse Primer | 5′-CCTTTTTTGCCCCATAGGAA-3′ | |
| TNF-α | Forward Primer | 5′-AGCCCCCAGTCTATATCCTT-3′ |
| Reverse Primer | 5′-CTCCCTTTGCAGAACTCAGG-3′ | |
| GAPDH | Forward Primer | 5′-ACCCAGAAGACTGTGGATGG-3′ |
| Reverse Primer | 5′-CACATTGGG GGTAGGAACAC-3′ |
Statistical analysis All data from this study were obtained from two independent experiments, carried out in triplicate, and expressed as mean ± standard deviation (SD). Statistical differences were analyzed using the ANOVA and Tukey multiple comparison test to compare between different groups. Differences were considered significant when p < 0.05. Statistical analysis was performed using the SPSS software package (SPSS 22 for Windows, SPSS Inc., Chicago, IL, USA).
Effect of DFO extract on cell viability in RAW 264.7 cell line Effects of different concentrations of DFO on cell cytotoxicity was evaluated. The maximum concentration of DFO which yielded cell viability higher than 80% was chosen. This criteria have been applied in other studies (Matsuda et al., 2003; Sae-Wong et al., 2011; Soonthornsit et al., 2017). However, some studies have selected a concentration that yielded cell viability higher than 85–90% (Joo et al., 2014). For the LPS treatment in this study, the cell viability was determined from cells treated for 24 h with DFO at 0.5, 1.0 and 2.0 mg/mL. These three concentrations represented low, medium and high concentrations, respectively. A sample of FOS at a concentration of 2.0 mg/mL was also prepared and then stimulated with LPS (1 µg/mL) for 24 h. Control samples consisted of RAW 264.7 cells alone (negative control denoted as vehicle) and cells incubated with LPS without pre-treatment with DFO or FOS were regarded as the positive control. The results of cell viability are shown in Fig. 1. It was found that there were no significant differences between the control samples and those treated with DFO or FOS. The effect of the prebiotic on PGE2, NO and TNF-α production was further investigated. These enzymes play important roles in a variety of inflammatory conditions (Sharma et al., 2007) and the inhibition of these inflammatory mediators is crucial in the prevention of inflammatory diseases.
Effect of DFO on cell viability in LPS-induced RAW 264.7 cells. Note: Cells were treated with various concentrations of DFO and FOS (mg/mL) for 24 h, then stimulated with LPS at a concentration of 1 mg/mL for 24 h. Treatments with different letters are significantly different (P < 0.05).
Effect of DFO extract on PGE2, NO and TNF-α production Many studies have revealed the role of PGE2 in metabolizing arachidonic acid and as a significant mediator of the inflammatory process (Meram and Wu, 2017). PGE2 is synthesized by PG G/H synthase and the COX enzyme with the stimulation of LPS or cytokines (McAdam et al., 1999; Hwang et al., 2019).
The effects of the prebiotic compounds (FOS and DFO) on PGE2 production in the culture medium of the LPS-induced RAW 264.7 cells were determined using ELISA assay. As shown in Fig. 2A, the lowest amount of PGE2 was produced in the control sample consisting of RAW 264.7 cells (vehicle: 4.11 ± 5.59 pg/mL). The LPS-stimulated macrophage control group released PGE2 at the highest level of 245.94 ± 44.96 pg/mL. Meanwhile, lower levels of PGE2 were observed in all the cells treated with FOS and DFO, while cells treated with DFO resulted in a dose-dependent decrease of PGE2 production in the LPS-stimulated RAW 264.7 cells. Interestingly, cells exposed to the same concentration of FOS and DFO (i.e., the maximum concentration of 2.0 mg/mL) produced significantly (p < 0.05) different values for PGE2. Lower PGE2 released was observed with DFO (51.77 ± 0.98 pg/mL), while FOS exhibited higher PGE2 value (134.76 ± 3.18 pg/mL).
Effects of DFO on the production of PGE2 (A), NO (B) and TNF-α (C) in culture medium of LPS-induced RAW 264.7 cell line. Notes: Vehicle: Microphage RAW 264.7 cells with no treatment (negative control); LPS: RAW 264.7 incubated for 24 h with LPS without prebiotic pretreatment (positive control); FOS 2.0 + LPS: Microphage pre-treated with FOS for 24 h at 2 mg/mL and stimulated with LPS for 24 h; DFO 0.5, 1.0, and 2.0 + LPS: Microphage pretreated with DFO for 24 h at 0.5, 1 and 2 mg/mL and stimulated with LPS for 24 h. The different letters are significantly different (p < 0.05).
The results on the effects of the prebiotic compounds on NO production in a culture medium of LPS-induced RAW 264.7 cells are shown in Fig. 2B. The lowest amount of NO was produced in the negative control sample (0.00 ± 0.04 µM), while the LPS-stimulated macrophage positive control sample released significantly (p < 0.05) more NO (60.25 ± 4.59 µM) than any of the other groups. Interestingly, the released NO was lower in all the cells treated with DFO, while cells treated with DFO resulted in a dose-dependent decrease in NO production in the LPS-stimulated RAW 264.7 cells. Additionally, when cells were exposed to the same concentration (2.0 mg/mL) of DFO and FOS, lower NO production was observed in cells treated with DFO than in those treated with FOS.
Another key mediator in the inflammatory process are cytokines. They are low-molecular-weight soluble proteins mainly produced through transcriptional and translational regulation upon external stimulation. TNF-α is pro-inflammatory cytokine which can induce inflammation and inhibit tumor genesis. The main role of TNF-α is the regulation of inflammatory and immune responses by regulating immune cells (Shokryazdan et al., 2017). The results on the effects of FOS and DFO on TNF-α production in a culture medium of LPS-induced RAW 264.7 cells are shown in Fig. 2C. The lowest amount of TNF-α was produced by the untreated macrophage control samples (289.28 ± 37.75 pg/mL), while the LPS-stimulated macrophage positive control group released TNF-α at the highest level of 4002.81 ± 129.76 pg/mL. Lower release of TNF-α was observed in all the cells treated with the prebiotic compounds. However, cells exposed to the minimum concentration of DFO produced the strongest inhibition of TNF-α production, which was significantly different from the positive control. Furthermore, the release of TNF-α by the cells exposed to the same concentration (2.0 mg/mL) of FOS and DFO, exhibited similar values as 3679.08 ± 175.10 pg/mL and 3813.26 ± 269.53 pg/mL), respectively.
Effect of DFO extract on COX-2, iNOS and TNF-α mRNA expression in RAW 264.7 cell line The levels of expression of COX-2, iNOS and TNF-α mRNA were examined by real-time PCR analysis to evaluate whether they were correlated with the reduced production of PGE2, NO and TNF-α as described above. The mRNA expression of COX-2, iNOS and TNF-α were noted to be significantly (P < 0.05) higher in the LPS positive control group than in the negative control group consisting of RAW 264.7 cells were then investigated.
The mRNA expression of COX-2 is shown in Fig. 3A. From the results, all the samples treated with LPS including those pre-treated with all concentrations of the prebiotics significantly (P < 0.05) produced higher COX-2 expression than the negative control group. However, the DFO at concentrations of 0.5 and 1.0 mg/mL had significantly (P < 0.05) decreased COX-2 expression compared to the LPS positive control. For samples pre-treated with FOS and DFO at a concentration of 2.0 mg/mL, COX-2 expression was not significantly different from that of the LPS group. Nonetheless, they were significantly (p < 0.05) higher compared to those pre-treated with lower concentrations of DFO.
Effects of DFO on the mRNA expression of COX-2 (A), iNOS (B) and TNF-α (C) in LPS-induced RAW 264.7 cell line. Notes: Vehicle: Microphage RAW 264.7 cells with no treatment (negative control); LPS: RAW 264.7 incubated for 24 h with LPS without prebiotic pretreatment (positive control); FOS 2.0 + LPS: Microphage pretreated with FOS for 24 h at 2 mg/mL and stimulated with LPS for 24 h; DFO 0.5, 1.0, and 2.0 +LPS: Microphage pretreated with DFO for 24 h at 0.5, 1 and 2 mg/mL and stimulated with LPS for 24 h. The different letters are significantly different (p < 0.05).
The mRNA expression of iNOS is shown in Fig. 3B. According to the results, DFO at a concentration of 0.5 mg/mL produced the lowest reduction (1.6 folds) of iNOS expression. Whereas there were no significant differences in iNOS expression between other groups given prebiotic pretreatment. In contrast, DFO at a concentration of 1 mg/mL showed significantly (P < 0.05) higher expression of iNOS than the group pretreated with 0.5 mg/mL of DFO. This could be interpreted that the inhibition of iNOS expression in this study was not dose dependent.
The mRNA expression of TNF-α as illustrated in Fig. 3C, shows that after treatment with FOS and DFO at a concentration of 2.0 mg/mL, TNF-α expression significantly (P < 0.05) increased compared to both the 0.5 and 1.0 mg/mL DFO groups and the LPS positive control group. However, there were no significant differences in the expression of TNF-α between the 0.5 and 1.0 mg/mL DFO groups and the LPS group. Thus, the expression of TNF-α at a concentration of 2.0 mg/mL of DFO was significantly higher compared to lower concentrations.
In this study, the expression level of PGE2 and TNF-α, and NO, and the upstream transcription levels of COX-2, TNF-α, and iNOS were determined. These molecules are crucial mediators in the inflammatory process that were generated in cells and secreted in a culture medium of RAW 264.7 macrophage cells.
Several studies have reported using RAW 264.7 macrophage cell as a model organism for evaluating the anti-inflammatory activity of food compounds (Zhou et al., 2015; Cheong et al., 2018; Oliveira et al., 2019). In this study, LPS was used to induce inflammatory markers since murine macrophages recognize LPS via specific receptors, especially Tolk-like receptor 4 (TLR4), and trigger the transcription factor NF-κB, leading to increased production inflammatory markers, such as NO, iNOS, PGE2, COX-2, and pro-inflammatory cytokines such as TNF-α and IL-6. These inflammatory markers perform key roles in the regulation of inflammatory responses (Meram and Wu, 2017). It was observed that DFO showed no cytotoxicity toward the RAW 264.7 macrophage cells up to a concentration of 2000 µg/mL but supported their growth. This result indicates that the anti-inflammatory effect of DFO in LPS-induced RAW 264.7 macrophages was not a false positive due to cytotoxicity. Moreover, we have done a preliminary study of DFO effect on NO production from RAW 264.7 culture medium with Griess reaction the results revealed that the single effect of various concentrations (0.5–2 mg/mL) of DFO could not induce NO production by Griess reaction. We observed that the single effect of DFO produced nitric oxide in the range of 0.0–3.3 µM but no significant difference compared to the vehicle group. Therefore, these responses indicate that the single effect of DFO did not display any immunomodulatory activity on NO production in macrophages cell.
The anti-inflammatory activity of DFO demonstrated the ability to attenuate the production of important inflammatory mediators such as NO and PGE2 and the expression of COX-2. The production of PGE2 and expression of COX-2 were significantly decreased. From this result, it is possible that DFO may act in a similar manner to non-steroidal anti-inflammatory drugs, by inhibiting cycloxygesnase, especially COX-2 (Chaiamnuay et al., 2006; Liu et al., 2017). Moreover, when comparing the use of oligosaccharides at the same concentration, DFO revealed a better efficacy in inhibiting NO production than commercial FOS. This beneficial effect was associated with a reduction in the production and/or expression of different inflammatory mediators that have been previously reported in other studies relating to oligosaccharides from different natural sources. Previous studies have shown anti-inflammatory activities in LPS-induced RAW 264.7 cells, for example, NAOS derived from red algae (Wang et al., 2017), guluronate oligosaccharides obtained from alginate (Zhou et al., 2015), oligosaccharide from Leuconostoc lactis (Lee et al., 2018), chitosan oligosaccharides (Yang et al., 2010), and xylo-oligosaccharides from xylan (Chen et al., 2012). Their effects may be associated with the interactions of different oligosaccharides such as FOS, inulin, and MOS with carbohydrate receptors (mannose receptors, galectin family receptors, and toll-like receptors) positioned on either macrophages or immune cells in the gastrointestinal tract (Seifert and Watzl, 2007). They may also reduce inflammatory responses by down-regulating mitogen-activated protein kinase and nuclear factor kappa B (NF-κB) signaling pathways in LPS-stimulated macrophages (Wang et al., 2017; Cheong et al., 2018). In addition, the degree of polymerization-4 of NAOS may significantly reduce the production of pro-inflammatory cytokines, such as TNF-α and IL-6, and release NO in LPS-induced macrophages (Wang et al., 2017). Similarly, Tan et al. (2018) reported that the bond between TLR and POS regulated the NF-κB and COX-2 signaling pathways in the expression of inflammatory cytokines (TNF-α, interleukines and PGE2).
On the other hand, the data from the present study revealed that the expression of iNOS was reduced only at a lower concentration (0.5 mg/mL) of DFO pre-treatment on the macrophage cells. However, the group pre-treated with a higher concentration showed no inhibition of the transcription and translation level, nor the expression of TNF-α. These could be due to the fact that NO and TNF-α in macrophage cells may play a role in the resolution of inflammation (Shaw et al., 2005; Parameswaran and Patial, 2010). Also, the macrophages are the key immune cells in mediating both the acute inflammatory phase and the repair phase after tissue damage. They are also capable of switching from pro-inflammatory to anti-inflammatory cells, which sustain tissue repair and help in maintaining homeostasis (Watanabe et al., 2019). DFO might be binding to macrophage at Toll-like receptor (TLR) via fructan binding site. Several studies reported that FOS and inulin-type fructans act as signals in animals, stimulating immune cell activity through TLR mediated signaling (Vogt et al., 2013; Peshev and van den Ende, 2014; Song et al., 2014). Therefore, various concentrations of DFO containing various fructan DP might influence the interaction between fructan and TLR binding mode.
Our results have shown that there is a correlation between gene expression and cytokine released. We believe the reasons could be related to; 1) presence of a single inducing agent (LPS) which is not a continuous stimulation (high transcription and translation markers), and samples collected for gene cytokines tests had same endpoint of detection time. Hence, gene and cytokine expression did not follow the same trend but showed a contrasting result. 2) A detecting time at the same endpoint may cause the difference in gene expression and cytokines production level. This is because gene expression will occur at a higher amount before it is translated into cytokine production. Therefore, when detected at the same endpoint with single stimulation, the level of gene or cytokine might not show the same high expression at the same concentration of DFO pre-treated cells. Gene expression is lower in the transcriptional process, whereas production of some cytokines or inflammatory mediators is higher in the translational process. 3) DFO composition and appropriate concentration which affected the transcription and translation process revealed the correlation between gene expression and cytokine production levels. In terms of DFO composition, a complex form of DFO is composed of soluble carbohydrate (many degrees of polymerization of oligosaccharides as a major component (75%) but not polysaccharide), and other components such as carbohydrate (except oligosaccharides), ash (mineral) and protein. Hence, the results have shown the synergistic effect of the DFO composition. Moreover, other reviews on the single effect of plant polysaccharides and oligosaccharides on immunoregulation properties in RAW 264.7 macrophage cell found that the structure of these substances can influence different responses, including enhancing the production of cytokines. Activation by plant polysaccharides and oligosaccharides may cause many inflammatory mediators secreted in macrophage which act as immoderate inflammation in the body (Yin et al., 2019). However, this effect may involve the number of inflammatory mediators. For example, the suitable amount of nitric oxide (NO) released by macrophage can exert beneficial functions and prevent the body from harmful factors. In a situation where the macrophages receive continuous stimulation and receive excessive NO, this may lead to sepsis and local or systemic inflammatory disorders (Yin et al., 2019). Similarly, the study of Mendis and team (2019) reported that arabioxylo-oligosacharides (AXOS) which contained substitution could decrease NO production in macrophages. Arabioxylans (AX) polysaccharides were inducers of NO production, acting as immunostimulatory. However, xylo-oligosaccharides (XOS) did not display any immunomodulatory activity with respect to NO production, except for some substitutions (Mendis et al., 2019). According to the fibril hypothesis, the fibrillar polysaccharides (large carbohydrate) are capable of binding to several pattern recognition receptors (PRR) molecules on the cell surface and can activate the immune response. Similarly, soluble short oligosaccharides (smaller carbohydrate) molecules are recognized by carbohydrate-binding domain of a single receptor molecule, deprived of activating an immune response (Latge, 2010). Investigation on pure components of DFO extract using inhibitors will be conducted to confirm the mechanism of DFO on the anti-inflammation effect in future studies. In terms of various concentrations of DFO treatment, we discovered that a lower concentration (0.5 mg/mL) of DFO was able to reduce the expression of COX-2 and iNOS, but could not reduce TNF-α. Furthermore, a higher concentration (2.0 mg/mL) of DFO was able to reduce the production of PGE2 and NO, but could not reduce TNF-α. From the result, it could be assumed that a low concentration of DFO preserved the isotonic condition of the cultured cells. Also, DFO has ability of penetrating into the cell to inhibit inflammatory markers at the transcription level. However, higher concentration of DFO results into change in the isotonic condition of cultured cells as they protect themself from DFO. Higher concentrations of DFO could not penetrate the cell, and subsequently, could not inhibit the transcription biomarker of TNF-α but DFO remained in cell culture medium to inhibit PGE2 and NO protein. Also, the DFO outer cell stays to inhibit inflammatory mediators at a translation level, which could indicate that DFO may function as an immunoregulator in the transcriptional process at a lower concentration. In contrast, higher concentrations of DFO could inhibit the translational process of an inflammatory response. Although, the effect of DFO has not been fully addressed, the data from the present study suggests that a lower concentration of DFO (0.5 mg/mL) provides a protective effect against pro-inflammatory cytokines and inflammatory processes.
This process is associated with the membrane receptors of macrophage cells through the suppression of pro-inflammatory responses via the PGE2/COX-2 and NO/iNOS pathways and the NF-κB signaling pathway. These pathways control the expression of genes and pro-inflammatory cytokines, as well as inflammation-inducing enzymes, adhesion molecules, and immune receptors such as IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, iNOS, COX-2 and TNF-α (Chen et al., 2018; Feng et al., 2018).
The results from this study demonstrate that oligosaccharides from dragon fruit (Hyloceus undatus) at a lower concentration of 0.5 mg/mL and at a higher concentration of 2.0 mg/mL appear to exhibit anti-inflammatory effects on RAW 264.7 macrophage cells through the PGE2/COX-2 and NO/iNOS pathways, except for TNF-α pathway as illustrated in Fig. 4. Nonetheless, the relevant mechanism needs to be confirmed in further in-depth studies for its effect on in vivo animal models before it is applied as a medical supplement in patients.
Dragon fruit oligosaccharide (DFO) effect in various concentrations on anti-inflammatory mechanism. Notes: LPS: lipopolysaccharide; COX-2: cyclooxygenase-2; PGE2: prostaglandin-E2; iNOS: inducible nitric oxide synthase; NO: nitric oxide; TNF-α: tumor necrosis factor-alpha; DFO 0.5: low concentration (0.5 mg/ml) of DFO; DFO 2.0: high concentration (2.0 mg/ml) of DFO; 
: inhibition effect; 
: reduction of inflammation.
Acknowledgements The authors acknowledge the financial support of Prince of Songkla University (Grant Number PHY600429S), and a PSU-Ph.D. Scholarship.
