Bioactive Phytochemicals from Mulberry: Potential Anti-Inflammatory Effects in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

The fruits of the mulberry tree (Morus alba L.), known as white mulberry, have been consumed in various forms, including tea, beverages, and desserts, worldwide. As part of an ongoing study to discover bioactive compounds from M. alba fruits, the anti-inflammatory effect of compounds from M. alba were evaluated in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Phytochemical analysis of the ethanol extract of the M. alba fruits led to the isolation of 22 compounds. Among the isolated compounds, to the best of our knowledge, compounds 1, 3, 5, 7, 11, 12, and 14–22 were identified from M. alba fruits for the first time in this study. Inhibitory effects of 22 compounds on the production of the nitric oxide (NO) known as a proinflammatory mediator in LPS-stimulated RAW 264.7 macrophages were evaluated using NO assays. Western blot analysis was performed to evaluate the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5). We evaluated whether the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5) following LPS stimulation in RAW 264.7 macrophages occurred because of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKβ), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB) and activations of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Cyclo(L-Pro-L-Val) (5) significantly suppressed phosphorylations of IKKα, IKKβ, IκBα, and NF-κB and activations of iNOS and COX-2 in a concentration-dependent manner. Taken together, these results indicate that cyclo(L-Pro-L-Val) (5) can be considered a potential therapeutic agent for the treatment of inflammation-associated disorders.


Introduction
Inflammation is a complex physiological response to foreign harmful stimuli including particles, toxic compounds, bacteria, and viruses. In the host defense system of macrophages, the regulation of inflammation is involved in immune function [1]. In response to lipopolysaccharide (LPS), macrophages overproduce proinflammatory mediator nitric oxide (NO) and cyclooxygenase (COX-2) to upregulate inflammatory states. NO is synthesized by inducible nitric oxide synthase (iNOS) via the NF-kB signaling pathway. Therefore, inhibition of LPS production and related mechanisms are considered useful targets in research to discover potential anti-inflammatory compounds [2].
The fruits of mulberry tree (Morus alba L.) belonging to the family of Moraceae are known as white mulberry, which is popularly consumed in various forms, including tea, beverages, and desserts, worldwide [3]. In Korea and China, where this plant is harvested, the leaves of M. alba have been traditionally used as a fodder for silkworms, as well as a medicinal herb to treat diabetes and improve eyesight [4]. The pharmacological activities of M. alba have also been actively studied, with reports that the white mulberry has a number of beneficial effects such as antioxidant [5][6][7], anti-microbial [8], anti-obesity [9,10], and anti-inflammatory activities [7].
As part of a continuing research project to discover bioactive natural products, our group has investigated biologically active and structurally interesting compounds from the ethanol extract of fruits of M. alba [7,[11][12][13][14]. In our previous study, we observed that butyl pyroglutamate identified from the fruits of M. alba exhibits protective effects against apoptosis, characteristic of cisplatin-induced kidney cell damage [11]. Our previous research has also shown that indole acetic acid derivative in M. alba induces apoptosis via both death receptor-mediated extrinsic pathway and mitochondria-mediated intrinsic pathway [12]. Odisolane, a new oxolane derivative identified from M. alba, was found to inhibit the tube formation of human umbilical vein vascular endothelial cells via vascular endothelial growth factor-mediated mechanistic pathway [13]. In addition, loliolide in M. alba was demonstrated by our group to protect INS-1 human pancreatic β-cells against apoptosis triggered by streptozotocin [14]. In particular, our recent research on ethanol extract from mulberry fruits reported its inhibitory effect on LPS-stimulated NO production and iNOS expression in a mouse macrophage cell line (RAW 264.7), suggesting its potential for anti-inflammatory effect [7]. In the present study, the ethanol extract of M. alba fruits was further investigated to identify potential anti-inflammatory compounds. Phytochemical analysis of the extract of M. alba fruits led to the identification of 22 compounds . Their structures were determined by detailed analysis of their nuclear magnetic resonance (NMR) spectroscopic and physical data as well as mass spectrometry (MS) data from liquid chromatography-mass spectrometry (LC-MS) analysis. Herein, we report the isolation and structural identification of the compounds  and their anti-inflammatory potential in RAW 264.7 cells and basic underlying mechanism of action.

Effects of Compound 5 on the LPS-Induced Expression of IKKα/β, I-κBα, and NF-κB in RAW 264.7 Mouse Macrophages
To confirm that compound 5 might be correlated with the inhibition of protein expression of IκB kinase alpha and beta (IKKα/β), inhibitor of kappa B alpha (I-κBα), and nuclear factor kappa B (NF-κB), we performed Western blot analysis. When the RAW264.7 cells were stimulated with LPS (1µg/mL) for 24 h, the protein expression of IKKα/β, I-κBα, and NF-κB was increased. However, treatment with compound 5 at 50 µM and 100 µM significantly inhibited expression of IKKα/β, I-κBα, and NF-κB in a concentration dependent manner ( Figure 4).

Effects of Compound 5 on the LPS-Induced Expression of iNOS and COX-2 in RAW 264.7 Mouse Macrophages
As a result of conducting a follow-up experiment based on the above results, we found that the protein expression of iNOS and COX-2 was increased after stimulation with LPS (1µg/mL) for 24 h, whereas treatment with compound 5 at 50 µM and 100 µM significantly inhibited this overexpression in a concentration dependent manner in RAW264.7 cells ( Figure 5).

Discussion
Many studies have been performed with respect to the anti-inflammatory activity of extract of the mulberry fruit (Morus alba L.) [7,[34][35][36][37]. In contrast, studies on which compounds in mulberry fruit have anti-inflammatory activity are still lacking.
In our previous study, the anti-inflammatory activity of the ethanol extract of the mulberry fruit was reported [7]. As an extension of previous research, our present study performed phytochemical analysis to isolate 22 chemical constituents from the ethanol extract and their non-toxic concentrations were investigated in RAW 264.7 macrophages. In addition, the inhibitory effect on LPS-stimulated NO production and its underlying molecular mechanism were investigated in RAW 264.7 macrophages. Kang et al. recently reported the inhibitory effect of cyclo(L-Pro-D-Val) on LPS-induced endothelial inflammatory responses [38], but they used different cells from ours, and the related mechanistic studies have not yet been conducted. In the present study, the anti-inflammatory action of cyclo(L-Pro-L-Val) and its mechanism of action were evaluated. These results could be a potential scaffold for the development of therapeutic agents to treat inflammatory disorders.
During the inflammatory response to LPS, RAW 264.7 macrophages play a central role in a regulating overproduction of a pro-inflammatory mediator, NO, in cell-based models of inflammation [39]. In this study, compound 5 markedly inhibited the NO production in a concentration-dependent manner in LPS-treated RAW 264.7 cells. Interestingly, its inhibitory effect was similar to that of L-NMMA. L-NMMA is an inhibitor of NO synthesis [40]. There was a report that evaluated the inhibitory effect of L-NMMA on expressions of IKKα/β, I-κBα, NF-κB, and iNOS in LPS-treated RAW 264.7 cells [41].
In response to LPS, IKK composed of two catalytic subunits (IKKα and IKKβ) are phosphorylated [42]. Phosphorylation of both IKKα and IKKβ leads to IκB phosphorylation, which directly contributes to activation of a nuclear transcription factor, NF-κB [43]. NF-κB regulates the transcription of iNOS and COX-2 by binding to specific DNA sequences. Induction of iNOS and COX-2 produces NO and prostaglandin E2 (PGE2), respectively [44]. Thus, we investigated whether compound 5 could inhibit expressions of IKKα/β, I-κBα, NF-κB, iNOS, and COX-2 in LPS-treated RAW 264.7 cells using Western blot analysis. Our studies demonstrate that compound 5 in a concentration-dependent manner inhibited LPS-mediated overexpressed IKKα/β, I-κBα, and NF-κB in RAW264.7 cells. In addition, increased expression of the iNOS and COX-2 was observed in response to LPS stimulation which was inhibited after treatment with compound 5. These results indicated that compound 5 inhibited the expression of iNOS and COX-2 via inhibition of NF-κB/I-κBα pathway, thus lower expression of iNOS resulted in lower NO production ( Figure 5). Although more experiments including animal research are needed to clarify effect bioavailability and bio-accessibility, compound 5 has potential as a favorable candidate for the treatment of inflammatory diseases.

Plant Material
The fruits of M. alba were collected in China in January 2014. A voucher specimen (MA 1414) of the material was identified by one of the authors (K.H. Kim) and was placed in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Measurement of NO Produced by RAW 264.7 Cells
RAW 264.7 cells (3 × 10 4 cells/well) were exposed to the indicated concentrations of compounds 1-22 for 1 h and then incubated for an additional 24 h with LPS (1 µg/mL). At the end of the incubation, each culture supernatant was blended with the Griess reagent to determine NO production by RAW 264.7 cells. Optical density at 540 nm of the mixture was determined using a spectrophotometer microplate (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA).

Western Blot Analysis
RAW 264.7 cells (4 × 10 5 cells/well) were exposed to the indicated concentrations of compound 5 for 1 h, and then incubated for an additional 24 h with LPS (1 µg/mL). At the end of the incubation, the RAW 264.7 cells were lysed with lysis buffer (Cell Signaling Technology, Beverly, MA, USA), supplemented with 1 mM phenylmethylsulfonyl fluoride, for 20 min. For Western blot analysis, 20 µg of the total protein from the cell lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electro-transferred to a polyvinylidene fluoride (PVDF) membrane. Each PVDF membrane was probed with primary antibodies (Cell Signaling Technology, Beverly, MA, USA) overnight, then incubated with horse radish peroxidase-conjugated anti-rabbit antibodies (Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature, and visualized using an enhanced chemiluminescence detection reagent (GE Healthcare, Little Chalfont, UK). Western blot signals were detected by FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Statistical Analysis
All assays were performed in triplicate and repeated at least three times. All data are presented as the mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) and multiple comparisons with the Bonferroni correction. A p value of <0.05 indicated statistical significance. All analyses were performed using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA).

Conclusions
In summary, as part of an ongoing research project to discover bioactive natural products [45][46][47][48][49][50], phytochemical examination of the extract of M. alba fruits led to the isolation and identification of 22 compounds in the process of discovery of potential anti-inflammatory compounds. This study demonstrates that compound 5 inhibited NO production and iNOS and COX-2 in LPS-stimulated RAW 264.7 macrophages. At least in part, these inhibitory effects are mediated via inhibition of NF-κB/I-κBα pathway ( Figure 6). Thus, these findings supported the utilization of cyclo(L-Pro-L-Val) (5) as a favorable candidate for the treatment of inflammatory diseases. Figure 6. Schematic illustration of the mechanism for the potential role of compound 5 isolated from M. alba fruits in inflammatory responses. LPS, lipopolysaccharide; p, phosphorylated; IKK, IκB kinase alpha; IκBα, inhibitor of kappa B alpha; p56 and p50, cellular proteins; MAPK, mitogenactivated protein kinase; Ac, activated; TNF-α, tumor necrosis factor alpha; IL-6, interleukin 6; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase.