Anti‐inflammatory and anti‐insulin resistance activities of aqueous extract from Anoectochilus burmannicus

Abstract This study investigated biological activities including antioxidative stress, anti‐inflammation, and anti‐insulin resistance of Anoectochilus burmannicus aqueous extract (ABE). The results showed abilities of ABE to scavenging DPPH and ABTS free radicals in a dose‐dependent manner. Besides, ABE significantly reduced nitric oxide (NO) production in the lipopolysaccharide (LPS)‐treated RAW 264.7 via inhibition of mRNA and protein expressions of nitric oxide synthase (iNOS). The LPS‐induced mRNA expressions of cyclooxygenase‐2 (COX‐2) and interleukin 1β (IL‐1β) were suppressed by ABE. Moreover, ABE exerted anti‐insulin resistance activity as it significantly improved the glucose uptake in tumor necrosis factor (TNF)‐α treated 3T3‐L1 adipocytes. In addition, ABE at the concentration of up to 200 μg/mL was not toxic to human peripheral blood mononuclear cells (PBMCs) and did not induce mutations. Finally, the results of our study suggest the potential use of A. burmannicus as anti‐inflammatory, anti‐insulin resistance agents, or food supplement for prevention of chronic diseases.

Several physical and chemical stimulants are factors involved in the inflammatory process such as free radicals which cause oxidative stress (Li & Wang, 2011). During an inflammatory response, excessively high levels of oxidative stress often result in cellular damage by initiating chemical chain reactions such as lipid peroxidation or the oxidation of DNA and proteins leading to cellular dysfunction (Agarwal, Saleh, & Bedaiwy, 2003;Lobo, Patil, Phatak, & Chandra, 2010) and cell death (Lefebvre et al., 2002).
Macrophage plays important roles in the autoregulatory loop of the inflammatory process (Shibata et al., 1998). Lipopolysaccharide (LPS) can activate toll-like receptor 4 (TLR4) in macrophages to induce the expression of many inflammatory genes causing the production of several inflammatory mediators such as nitric oxide (NO), prostaglandins (PGs), and many proinflammatory enzymes (Creely et al., 2007). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are directly responsible for elevated levels of NO and PGs, respectively, leading to promotion of pathological inflammation (Cho, Cho, & Song, 2005;Huang et al., 2013). Moreover, NO has effect on COX-2 activity (Li & Wang, 2011;Salvemini et al., 1993).
In recent years the researchers have tried to substantiate antiinflammatory and anti-insulin resistance properties of many natural products. It is noteworthy that effective therapeutic agents on controlling a variety of inflammatory diseases mostly target macrophages and their products (Fujiwara & Kobayashi, 2005). Many studies have extensively focused on anti-inflammation and metabolic syndrome.
For example, anti-TNF-α blockade can improve insulin resistance in rheumatoid arthritis patients (Gonzalez-Gay et al., 2006). Interleukin-1-receptor antagonist has been reported to increase insulin sensitivity in type 2 diabetes mellitus patient (Akash, Shen, Rehman, & Chen, 2012). Thus, studies of anti-inflammation have become a main target for prevention and treatment of obesity-related insulin resistance.
In this study, we focused on biological effects of A. burmannicus, which is found in the northern part of Thailand. We aimed to determine whether A. burmannicus aqueous extract exerts antioxidative stress, anti-inflammation, and anti-insulin resistance. Aqueous extract of ABE was prepared and its phenolic content determined. Biological properties were examined including antioxidative stress and anti-inflammation in LPS-induced RAW 264.7 macrophage cells and anti-insulin resistance in TNF-α-induced 3T3-L1 adipocytes. The knowledge from this study would provide supportive evidences for implementation and development of this plant as an alternative medicine or functional food in order to prevent and/or treat chronic diseases including type 2 diabetes mellitus which could be caused by inflammation.

| Preparationofplantextracts
Plant materials and extraction were cultivated by tissues culture engineering (Queen Sirikit Botanic Garden, Chiang Mai, Thailand). Dried whole plant of A. burmannicus was soaked in diH 2 O and subjected to autoclave at 121°C for 30 min according to the method of Kim & Jang, (2011) with slight modifications. Then, the aqueous fraction was filtered and lyophilized to obtain crude ABE powder.

| Determinationoftotalphenoliccompounds
Total phenolic content of ABE was determined according to the method of by Subedi et al., (2014) with slight modifications. Briefly, 300 μL of the extracts was added to 400 μL of 10% equivalent Folin-Ciocalteu reagent and incubated for 3 min in dark, room temperature.
Then 300 μL of 7.5% equivalent/L Na 2 CO 3 was added and the mixture was allowed to stand for 20 min in dark, after which absorbance readings at 765 nm was measured using a spectrophotometer. The phenolic content was calculated using the linear equation based on the calibration curve and expressed as the total phenolic contents equivalence (CE)/g dry weight.

| Determinationofphenolicderivatives contentinABE
Phenolic compounds were determined by HPLC using a C18 column (250 mm × 4.6 mm, 5 μm). Gradient elution was performed using two solvents; A (0.1% trifluoroacetic acid in water) and B (100% methanol) for determining phenolic compounds. Ten microliters of the 10 mg of ABE was dissolved in 1 mL of diH 2 O and injected into the column with a flow rate 1.0 ml/min and detected at 280 and 325 nm. Peak area and retention time of the extract sample were compared with standard curves of various concentrations of standard catechin, chlorogenic acid, coumaric acid, ferrulic acid, gallic acid, hydroxybenzoic acid, protocatecheuic acid, and vanillic acid.

| ABTSradicalscavengingassay
The ABTS + scavenging ability was determined according to the method of Jatinder Kumar et al., (2014). A mixture of 7 mmol/L ABTS + stock solution with 2.45 mmol/L potassium persulfate (1:1, v/v) was left in the dark at room temperature for 12-16 hr until the reaction was complete and the absorbance was stable. The ABTS + reagent was diluted with 95% ethanol to an absorbance of 0.700 ± 0.02 at 734 nm (Microplate reader Synergy Hybrid Reader, BioTek). An aliquot (10 μL) of each sample was mixed with 990 μL ABTS + reagent and the absorbance was read at 734 nm after 6 min at 30°C in the dark. A reference standard was Trolox. The antioxidant activity was defined as the IC 50 required to scavenging ABTS + radicals by 50%, which was calculated from a log-dose inhibition curve.

| DPPHradicalscavengingassay
DPPH radical scavenging activity was evaluated using the following method from a previous study (Wang, Wang, & Yih, 2008). Twenty microliters of ABE solution were mixed with 180 μL of diphenylp-picrylhydrazyl radical (DPPH) in ethanol and 20 μL of different concentrations of sample and then incubated for 20 min at room temperature with light protection. The remaining DPPH was measured by colorimetric at 517 nm using a microplate reader (Synergy Hybrid Reader). The antioxidant activity of sample was shown as the IC 50 required to scavenging DPPH radicals by 50%, which was calculated from an inhibition curve.

| Celllinesandcellculture
RAW 264.7 macrophage-like cell line was obtained from CLS-Cell Lines Service, Germany. The cell line was cultured in an ultralow attachment culture dish in DMEM with l-glutamine supplemented with 10% heatinactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin solution and maintained at 37°C in a 5% CO 2 humidified atmosphere CO 2 incubator (Thermo Scientific). The cells were subjected to the experiments or subculture when they reached confluence of 80%. 3T3-L1 adipocyte cells from ATCC (American Type Culture Collection) were cultured in DMEM with l-glutamine supplemented with 10% FBS and 1% penicillin/streptomycin solution and maintained at 37°C in a 5% CO 2 humidified atmosphere CO 2 incubator. After reaching confluence on day 2, 3T3-L1 preadipocytes were differentiated by inducing with 0.5 mmol/L 3-isobutyl-1-metylxanthine (IBMX), 0.5 μg/mL dexamethasone, 5 μg/ mL insulin, and 10% fetal ovine serum for 72 hr, followed by another 72 hr in the same medium without IBMX and dexamethasone. Complete cell differentiation was obtained by incubating the cells in DMEM containing 10% FBS for 7-14 days. Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-hypaque. The mononuclear cells were carefully collected and rinsed twice with ice-cold phosphatebuffered saline (PBS) pH 7.4 and resuspended in fresh RPMI medium.

| TreatmentprotocolofRAW264.7macrophages
RAW 264.7 macrophage cells were seeded in plates or dish for 24 hr.
Then, medium was removed and replaced fresh medium with or without different concentrations of the ABE (0-200 μg/ml). After 2 hr of the incubation, 1 μg/mL of LPS was added and further incubated for 24 hr. After the treatment, cell-free culture medium or cell pellets were collected for further studies.

| DeterminationofNOproduction
After the treatment, the cell-free culture medium was collected to measure the amount of nitric oxide using Griess reagent (Sigma Aldrich) as described in the manufacturer's protocol. The amount of nitrite present in the samples was calculated by means of a standard curve generated using serial dilutions of NaNO 2 in fresh culture medium.

| Cytokineproductiondeterminations
The levels of IL-6 and TNF-α in the culture medium of the treated cells were determined by sandwich Enzyme Link Immuno-Sorbent Assay (BioLegend's ELISA MAXTM Deluxe Set, CA). Briefly, 100 μL of supernatant after treatment was used and assayed according the manufacturer's protocol for the relevant ELISA kit.

| Immunoblotanalysis
After the treatment with cytokine, RAW 264.7 cells were collected and lysed with RIPA buffer containing protease inhibitors. The protein samples were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 3% BSA in tween-TBS at 4°C, and then probed with primary antibodies specific to COX-2 (Cell signaling, USA), iNOS (Merck, USA), or β-actin (Sigma, USA) overnight. The membrane was washed five times with 0.015% tween-TBS for 30 min, and was incubated with horseradish peroxidase (HRP)-conjugated IgG secondary antibody for 2 hr at room temperature. An enhanced chemiluminescence (ECL) system was utilized to detect the HRP signal. Equal protein loading was evaluated in each membrane, which was stripped and reprobed with anti-β-actin.
RAW 264.7 macrophage cells were plated overnight in 6-well plate and incubated with ABE for 2 hr, and then stimulated with LPS (1 μg/mL) for 24 hr. Total RNA was extracted using Trizol reagent (Invitrogen). First-strand cDNA was synthesized using 1 μg of total RNA with oligo (deoxythymidine) primers and Superscript II reverse transcriptase (Invitrogen). The target cDNA was subsequently used as the template for RT-qPCR amplification using for 5 min, 4°C for 1 min, and 42°C for 60 min. The level of target cDNA was normalized by the expression of GAPDH and then measured as relative expression to the LPS-treated control.

| Statisticalanalysis
All values were given as mean ± standard derivation (X ± SD) from triplicate samples of three independent experiments. Overall differences among the treatment groups were determined using one-way analysis of variance (ANOVA) by Prism 5.0 software. p < 0.05 is regarded as significance.

| EffectofABEonscavengingofDPPHand ABTSradicals
Antioxidant activities of ABE were determined using DPPH and ABTS assays as shown in Figure 1. ABE had ability to scavenge DPPH free radical up to 70% (p < 0.05) in a dose-dependent manner. The 50% inhibition concentration (IC 50 ) was 3.39 ± 0.23 mg/mL (Fig. 1A). The ABTS assay system is a decolorization technique in which the radical is generated directly and stable. In this assay, the ABE fraction exhibited the suppressive effect on ABTS radical with an IC 50 value of 0.38 ± 0.02 mg/mL (Fig. 1B). These results suggested that ABE exerted antioxidant property which likely provides protective effect on oxidative stress related to inflammation.

| EffectofABEongenomestabilityofDrosophila melanogaster
To assess the genotoxic properties of ABE, the in vivo assay involving  Table 1, 20 mmol/L urethane statistically induced DNA mutations (total spot), which is in line with a formerly report (Laohavechvanich et al., 2006). The negative control together with ABE did not provoke any type of mutations implying that ABE was not genotoxic, although the used dose of ABE (500 μg/mL) was even higher than that of cytotoxicity assay (Fig. 1C-E).

| EffectofABEonNOproductioninLPS-stimulatedRAW264.7macrophages
To assess the anti-inflammatory activity in vitro, we determined the inhibitory effect of ABE on NO production in LPS-treated RAW 264.7 cells. The NO release was measured as the accumulation of nitrite in the culture supernatant. Significantly, ABE inhibited the LPS-induced nitrite production in a dose-dependent manner by 10-20% as compared to LPS-treated group ( Fig. 2A).

| EffectofABEonLPS-inducediNOSandCOX-2 expressioninraw264.7macrophages
NO production is catalyzed by iNOS enzyme whose expression can be upregulated in the LPS-induced RAW macrophages. The inhibitory effect of ABE on LPS-induced NO production as shown earlier might be due to the reduction in iNOS synthesis. The expressions of iNOS at both protein and transcript levels were then detected using immunoblotting and RT-qPCR, respectively. The results found that ABE obviously decreases iNOS protein level (Fig. 3A), but slightly inhibits the LPS-induced iNOS mRNA expression (Fig. 3B). Thus, the inhibitory Statistical diagnoses using estimation of spot frequencies and confidence limits according to Frei and Würgler (1988) for comparison with deionized water; +, Positive; −, Negative; i, Inconclusive. Probability levels: α = β = 0.05. One-sided statistical test "m" is an increased mutation frequency compared with the spontaneous frequency (m times). ABE, Anoectochilus burmannicus aqueous extract. Furthermore, the effect of ABE on expression of another inflammatory gene, COX-2, was elucidated as shown in Figure 3B. ABE could slightly reduce COX-2 mRNA expression about 20%. Alteration of COX-2 expression may lead to the decreased production of prostaglandin E2 (PGE2), which also contributes to the progression of inflammation (Ricciotti & FitzGerald, 2011).

| EffectofABEonTNF-αinducedinsulin-resistant3T3L1adipocytes
Insulin resistance in adipocytes has been shown to correlate with inflammatory response, especially TNF-α leading to a low glucose uptake into the cells. ABE treatment significantly sensitized the insulin response in TNF-α-treated adipocytes as the glucose uptake level was 40% higher than that of the inflammation control (Fig. 4).

| DISCUSSION
In this study, we have firstly reported antioxidative stress, antiinflammatory, and anti-insulin resistance activities of A. burmannicus.
This plant has been commonly used in traditional Thai and Chinese medicines in form of decoction prepared by boiling herbs with water for 15-60 min. As to mimic the instruction for the use in daily life, we prepared the aqueous extract by boiling at 121°C for 30 min that following the method of Kim & Jang, (2011) with slight modifications.
However, it is interesting whether the aqueous extract prepared by infusion is also biologically as effective as boiling since it would be further developed for consuming this plant as a hot tea.
Free radicals are potential inflammatory mediator contributing cellular or tissue damage, which is considered in the downregulation of inflammatory response (Lobo et al., 2010). ABE showed an efficiency to scavenge DPPH and ABTS free radicals in a dose-dependent manner. Antioxidant activity of ABE might be involved with its antiinflammation and anti-insulin resistance.
Our results showed that ABE significantly inhibits the LPS-induced NO production in macrophages.
Inducible NO synthase (iNOS), a key enzyme in NO production, is highly expressed during inflammation (Sharma et al., 2007). Several phenolic compounds, such as gallic acid, vanillic acid, coumaric acid, and ferulic acid, have been shown to directly inhibit the iNOS gene and protein expressions leading to the decreased level of NO production (Wang & Mazza, 2002). We found that ABE could suppress both mRNA and protein expressions of iNOS in LPS-induced macrophages.
These results suggested that ABE may attenuate NO synthesis via the downregulation of iNOS expression at the transcription and translation levels. However, ABE did not alter the protein expression of COX-2; an inflammatory enzyme that catalyzes arachidonic acid into prostaglandins, which is inflammatory mediator and contributing inflammation (Ricciotti & FitzGerald, 2011). Several studies reported similar antiinflammation effects without the alteration of COX2. Kim, Hwang, & Park, (2014) showed chloroform layer of Actinidia arguta stems extract did not alter the COX-2 level, but it strongly reduced NO production, proinflammatory cytokines in both protein and mRNA expression levels, and inhibited activation of NF-κB and mitogen-activated protein kinase (MAPKs). Likewise, Park, Kwon, & Sung, (2009) reported aloin could reduce NO production and level of proinflammatory cytokines, but had no effect on COX-2 expression. Therefore, the reduction in NO production and IL-1β level, but not COX-2 expression by ABE might be sufficiently effective against inflammation.
IL-1β and IL-6 are important inflammatory cytokines secreted by macrophages. TNF-α also is a major mediator in the development of chronic inflammation in diabetes mellitus and rheumatoid arthritis (Popa et al., 2007). Several studies reported suppressive effect of plant extracts on expression of these proinflammatory cytokines. For example, grape powder extract could reduce both inflammation and insulin resistance mediated by TNF-α in adipocytes (Chuang et al., 2011). In addition, Sasa borealis leaves extract could improve insulin resistance by reducing cytokines secretion in obese C57/BL6J mice (Yang, Lim, & Heo, 2010). In our study, ABE could diminish the LPSinduced expressions of IL-1β and IL-6, but was unable to inhibit either the elevated TNF-α mRNA or protein expression in the activated macrophages. Similar to our results, lycopene (Feng, Ling, & Duan, 2010) and methanolic extract from edible mushrooms (Moro et al., 2012) inhibited the LPS-induced production of IL-6 and NO with decreased mRNAs of IL-1β, IL-6, iNOS, and NO without the effect on TNF-α. Significantly, lycopene attenuated the effects of LPS by inhibiting a key inflammatory pathway related to mitogen-activated protein kinase (MAPK), ERK1/2 and p38 MAPK, and NF-κB, but not JNK (Feng et al., 2010). This observation supports the hypothesis that ERK1/2 predominantly triggers the expression of IL-1β and IL-6, whereas JNK is the main transduction pathway of TNF-α, after LPS stimulation (Swantek, Cobb, & Geppert, 1997). It is possible that ABE may inhibit the LPS activation of MAPK and NF-kB pathways leading to the decrease in IL-1β and IL-6.
Obesity and adipogenesis could increase production and secretion of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 from macrophages into adipose tissues. These cytokines, especially TNF-α, highly induce an insulin resistance in adipocytes in which the insulin signaling is interrupted resulting in an impaired glucose uptake (Makki et al., 2013;Nieto-Vazquez et al., 2008;Rotter, Nagaev, & Smith, 2003). Although ABE had no effect on TNF-α production in the LPS-treated macrophages, we found that a low dose of ABE (50 μg/ml) could inhibit TNFα-induced insulin resistance in adipocyte as shown by the increased glucose uptake. The anti-insulin resistance of ABE could be directly useful for prevention and treatment of diabetes. Kinsennoside from A. roxburghii showed a significant hypoglycemic effect in streptozotocininduced diabetic rats. It ameliorated β-cells' damage caused by oxidative stress and NO, and also prevented weight loss in the diabetic animals (Zhang et al., 2007). In the study by Jian-Gang Zhang (2015), polysaccharose isolated from A. roxburghii exerted antioxidant effect on diabetic mice leading to an improvement of glucose and lipid metabolism, an increase in immune protection, and a decrease in oxidative stress, which might contribute to its antidiabetic effect (Zhang et al., 2015).
In summary, we have demonstrated antioxidation antiinflammation, anti-insulin resistance, as well as safety of ABE. The anti-insulin resistance effect of ABE in TNF-α-induced adipocyte could be due to the inhibition of macrophage and adipocyte inflammation.
Significantly, ABE was not toxic as determined by in vitro cytotoxic and in vivo mutagenesis assays, suggesting its safety for further development and examination in animal and clinical models. Finally, our study provides scientific information for development and application of A. burmannicus as folk medicine, food ingredient, or food supplement for prevention of inflammation-related chronic diseases. Further studies should be carried out to investigate the active components in ABE and to provide an insight into the underlying mechanisms of these effects.

CONFLICTOFINTEREST
We have no conflict of interest to declare.