Anti-inflammatory effect of neo-lignan isoamericanin A via suppression of NF-κB in liposaccharide-stimulated RAW 264.7 cells

Purpose: To investigate the potential anti-inflammatory effects of the seeds of Opuntina humifusa and its active constituents. Methods: The extract of O. humifusa seeds was tested for the inhibition of nitric oxide (NO) production in liposaccharide (LPS)-stimulated RAW 264.7 cells using Griess reagent. The active constituents were isolated using bioassay-guided isolation methods. The effects of the active constituent on NO, proinflammatory cytokines, nuclear factor-kappa B (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Results: The seed extract of O. humifusa significantly attenuated LPS-induced NO production in RAW 264.7 cells (p < 0.05). Bioassay-guided fractionation resulted in the isolation of isoamericanin A as an active constituent. Isoamericanin A reduced LPS-induced production of NO, iNOS, and proinflammatory cytokines (TNF-α and IL-6) in a concentration-dependent manner (p < 0.05). Furthermore, the effect was accompanied by decreased translocation of NF-κB from the cytosol to the nucleus and the decreased phosphorylation of IκB in the cytosol induced by LPS (p < 0.05). Conclusion: The seed extract of O. humifusa and its active constituent, isoamericanin A, have antiinflammatory effects in LPS-stimulated RAW 264.7 cells, suggesting that they have potentials as antiinflammatory agents.


INTRODUCTION
Opuntia humifusa Raf. (Cactaceae) is a cactus known as the eastern prickly pear. It is widely cultivated in the southern parts of Korea and is locally called Cheonnyuncho. The stems and fruits of O. humifusa are used as food worldwide in the form of juice or freeze-dried powder because they are rich in polyphenols, flavonoids, minerals, and dietary fiber. Traditionally, it has been used to treat diabetes, inflammation, and rheumatoid arthritis [1]. In addition, there are reports on its anti-cancer effects on diverse cancer cells including glioblastoma, cervical carcinoma, and gastric adenocarcinoma [2,3]. O. humifusa has antibacterial [4], and anti-fungal [5] effects, and increases insulin sensitivity [6]. The inhibitory effect of O. humifusa against LPSinduced inflammation has also been reported [7,8].
However, studies on the biological activities of O. humifusa focused mainly on the stems and fruits; the properties of its seeds have not been studied yet. Thus, in this study, the anti-inflammatory effects of O. humifusa seeds were examined and their active constituent was isolated using bioassay-guided fractionation. The antiinflammatory mechanisms of the active constituents were also elucidated.

Preparation of the seed extract and isolation of isoamericanin A
The seeds of O. humifusa were collected after removing the flesh of the fruits. The seeds (3 kg) were pulverized and extracted with 80% methanol (MeOH). The MeOH filtrate was concentrated under vacuum, yielding 150 g of MeOH extract. The MeOH extract was suspended in distilled water and sequentially partitioned into n-hexane (2.2 g), chloroform (6.7 g), EtOAc (7.2 g), n-BuOH (8.0 g) and water (19.3 g). Each fraction was dissolved in DMSO for the bioassay. The chloroform fraction was subjected to open column chromatography and eluted with chloroform-MeOH (100:0 to 1:1) to give 12 sub-fractions (OCC1~OCC12). The subfraction OCC5 (0.2 g) was further purified by semi-preparative HPLC and eluted with acetonitrile (ACN)-H2O (30 to 60% ACN gradient) to yield compound 1 (15.2 mg). Compound 1 was identified as isoamericanin A based on the NMR and MS data compared with previously published data [9].

Evaluation of cell viability
RAW 264.7 cells plated on 96-well plates (5×10 4 cells/well) were pre-treated with test samples for 1 h and incubated with LPS for an additional 24 h. Then, the cells were incubated with MTT (5 mg/mL) for 3 h followed by DMSO for 30 min after removing the media. The absorbance was measured at 570 nm (Molecular Devices, CA, USA). All measurements were done in triplicate and repeated at least three times.

Determination of NO production
The production of NO was determined using Griess reagent. The cells pre-treated with test samples were incubated with LPS for 24 h. Then, the supernatants were centrifuged at 12,000 rpm for 15 min and incubated with Griess reagent at 37 °C for 20 min in the dark. The absorbance was measured at 540 nm using a microplate reader.

Enzyme-linked immunosorbent assay (ELISA)
The supernatants were collected from RAW cells after treatment with LPS for 24 h in the presence or absence of isoamericanin A (1, 2, or 4 μg/mL), and the production of cytokines was evaluated using ELISA [10] employing the following capture antibodies: TNF-α and IL-6 (BD Biosciences, San Jose, CA, USA). The absorbance was measured at 405 nm using a microplate reader. Cytokines were quantified based on a standard curve.

Statistical analysis
All data are presented as mean ± SD (n = 3). One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was employed for comparisons of two or more groups. Differences were considered statistically significant at p < 0.05.

Inhibitory effect of O. humifusa seed extract on NO production
The possible cytotoxicity of the methanol (MeOH) extract of O. humifusa seeds was tested by MTT assay. As shown in the results, the MeOH extract of O. humifusa seeds did not decrease the viability of RAW cells at up to 20 μg/mL. However, 20 μg/mL MeOH extract reduced LPSinduced NO production by 50% compared to that in the LPS-treated control groups ( Figure 1). Thus, the MeOH extract was further partitioned by solvent polarity into four sub-fractions (nhexane, chloroform, ethyl acetate and water). The sub-fractions were subjected to the MTT assay to evaluate possible cytotoxicity. Three sub-fractions (chloroform, ethyl acetate and water) did not alter the viability of RAW cells, whereas the n-hexane fraction significantly reduced the viability (Figure 1 A). Thus, the nhexane fraction was excluded from further studies. Then, three sub-fractions (20 μg/mL) were evaluated for their effects on LPSstimulated NO production in RAW cells. The chloroform fraction significantly reduced NO production compared to that of the control group (Figure 1 B), whereas the ethyl acetate fraction exhibited only a weak inhibitory effect. Thus, the chloroform fraction was studied further to identify the active components.

Inhibition of NO production by isoamericanin A
To obtain active constituents in the chloroform fraction of the O. humifusa seed extract, bioassay-guided isolation was performed using silica gel column chromatography and semipreparative HPLC. Based on these results, isoamericanin A was isolated as an active constituent from the chloroform fraction (Figure 2  A). The possible cytotoxicity of isoamericanin A was determined by the MTT assay. At up to 4 μg/mL, isoamericanin A did not reduce the viability of RAW cells (Figure 2 B). Treatment of cells with isoamericanin A significantly attenuated LPS-induced NO production in RAW cells (Figure 2 B) in a concentration-dependent manner (1, 2, and 4 μg/mL). Notably, 4 μg/mL isoamericanin A reduced NO production by 60% compared to that in the LPS-treated group.

Inhibition of iNOS and pro-inflammatory cytokine expression by isoamericanin A
iNOS is an enzyme responsible for NO production in RAW cells. The possible change in iNOS expression was determined by western blot analysis. The cells treated with LPS significantly increased the expression of iNOS whereas pretreatment of cells with isoamericanin A prior to LPS treatment reduced the expression of iNOS in a concentration-dependent manner (Figure 3 A and B) compared to that in the LPS-treated group. In particular, 4 μg/mL of isoamericanin A decreased iNOS levels to close to the controlgroup level. This result is consistent with the inhibitory effects of isoamericanin A on NO production as described above.
In addition, the secretion of pro-inflammatory cytokines TNF-α and IL-6 by RAW cells was determined by ELISA. As shown in Figure 3 C and D, LPS treatment of RAW cells significantly increased the concentration of TNF-α and IL-6 in the supernatant whereas pre-treatment with isoamericanin A prior to LPS treatment significantly reduced the levels of cytokines in a concentration-dependent manner. Western blot experiments were performed using specific antibodies to elucidate the effect of isoamericanin A on LPS-induced NF-κB activation. As shown in Figure 4A, treatment of cells with LPS significantly increased the amount of NF-κB (p65 and p50) in the nucleus, indicating that LPS increased the translocation of NF-κB (p65 and p50) from the cytosol into the nucleus, whereas phosphorylated IκB (p-IκB) in the cytosol was significantly increased. However, the pre-treatment of cells with isoamericanin A prior to LPS treatment reversed the effects of LPS on NF-κB translocation and IκB phosphorylation. Isoamericanin A decreased the translocation of NF-κB p65 and p50 and reduced the amount of NF-κB p65 and p50 in the nucleus compared to LPS-treated control group. The treatment of cells with 4 μg/mL isoamericanin A decreased the levels of NF-κB in the nucleus to close to that of the DMSO-treated control groups. In addition, isoamericanin A inhibited the phosphorylation of IκB located in the cytosol and decreased the amount of p-IκB in a concentration-dependent manner.

DISCUSSION
The stems and fruits of O. humifusa have been widely used as food and traditional medicines due to their beneficial effects on diabetes, arthritis, and inflammation [1]; the related biological activities have been reported. For example, the MeOH extract of O. humifusa stems exhibit anti-nociceptive and antiinflammatory effects [8]. The stems of O. humifusa significantly reduce serum totalcholesterol and blood-sugar levels in streptozotocin-induced diabetic rats [11]. The fruit of O. humifusa reduces the morphological changes observed in asthma, and suppresses UVB radiation-induced skin degeneration and non-melanoma skin carcinogenesis due to their anti-inflammatory effects [12,13]. The beneficial effects of O. humifusa on inflammation are accompanied by high antioxidant effects due to high levels of vitamin C, polyphenols, and flavonoids present [14]. High levels of antioxidants in O. humifusa reduce the production of reactive oxygen species, decrease oxidative damage, and eventually alleviate oxidative stress in inflammation [15].
Conversely, reports regarding the biological activities of O. humifusa seeds are very limited. For example, the seed extract of O. humifusa alleviates osteoporosis [16] and decreases serum cholesterol and triglyceride contents [17] in ovariectomized rats because of the high polyphenol content in the seeds. Reports on the biological activities of O. humifusa are mostly limited to the plant's stems and fruits. Here, we report the anti-inflammatory effects of the seeds of O. humifusa and the isolation of the active constituent, isoamericanin A.
Isoamericanin A was first isolated from Phytolacca americana L. in 1987 [18]. Additionally, isoamericanin A was isolated from Brazilian Joannesia princeps [9] and barley tea [19]. Initially, isoamericanin A was identified as an inducer of prostaglandin I2 [18] or as an antioxidant constituent [19]. It was also reported to enhance choline acetyltransferase activity [20]. Other than the above, very few biological activities of isoamericanin A have been reported.
To the best of our knowledge, this is the first report regarding the potent anti-inflammatory effects of isoamericanin A against LPSstimulated RAW cells. Isoamericanin A effectively lowered the production of NO and proinflammatory cytokines, that was accompanied by the inhibition of NF-κB translocation. The NF-κB pathway plays crucial roles in human inflammatory diseases including rheumatoid arthritis, atherosclerosis, asthma, and inflammatory bowel disease [21][22][23]. The activation of NF-κB at inflammation sites increases the transcription of pro-inflammatory cytokines, chemokines, COX-2, and iNOS [24]. Therefore, NF-κB is a potential therapeutic target for inflammatory diseases.

CONCLUSION
The extract of O. humifusa seeds significantly reduces NO production in LPS-stimulated RAW cells, and isoamericanin A was identified as its active constituent. Isoamericanin A effectively lowers the production of NO as well as the expression of iNOS and pro-inflammatory cytokines. These effects are accompanied by decreased translocation of NF-κB from the cytosol to the nucleus and decreased phosphorylation of IκB in the cytosol induced by LPS. Thus, the seeds of O. humifusa and isoamericanin A have potential as antiinflammatory agents.