EVALUATION OF IN VITRO ANTI-INFLAMMATORY, ANTI-DIABETIC AND ANTI-LIPOGENIC ACTIVITY OF NATURAL POLYPHENOLIC EXTRACTS AND THEIR PURE CONSTITUENTS

The long-term goals and objectives of our group are to identify bioactive natural products relevant to human disease prevention and health promotion. To attain this goal, the objective of this thesis was to demonstrate that bioactive polyphenol-rich extracts exert positive health benefits beyond basic nutrition, thus impacting overall health and wellness. We believe that this project has immense scientific merit from a human health perspective and will be of great public impact given that consumers are seeking natural and organic choices for improving health. In this investigation, we have focused our attention on investigating the biological activities of two novel phenolic-rich preparations derived from curcumin and maple syrup for future nutraceutical applications. The work herein assessed the anti-inflammatory properties of novel standardized curcumin formulation (Longvida®) and phenolic-enriched maple syrup extract using a well-known in vitro model of inflammation, lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages. RAW 264.7 cells were co-treated with 50 ng/mL LPS and test extracts for 24 hours and then inflammatory markers were measured using gene and protein expression. The activation of nuclear factor-kappa B (NFκB) was measured using luciferase activity. Both polyphenolic extracts were able to inhibit the LPS stimulated inflammation by down-regulating the inflammatory markers via targeting nuclear factor-kappa B (NFκB) (a major pathway modulated in inflammation) transcriptional activity in murine macrophages. Next, the work herein studied the anti-hyperglycemic and anti-lipogenic properties of novel standardized nutraceutical grade phenolic rich extract, (MSX). Human HepG2 hepatoma cells were treated with MSX for 24 h. Glucose consumption, AMP activated protein kinase (AMPK) activation and its target gluconeogenic gene expression were measured. The glucose levels were reduced by MSX in the hepatocytes though AMPK activation. Subsequently, the anti-lipogenic effect of MSX treatment in mature differentiated 3T3-L1 murine adipocytes and human visceral adipocytes was evaluated. MSX treatment to mature adipocytes decreased lipid accumulation, compared to control, in both murine and human adipocytes. In 3T3-L1 adipocytes, this effect was associated with downregulation of adipo/lipogenic protein expression (e.g. PPARγ, Srebp1c). We also observed reduced mRNA expression of the pro-inflammatory mediators, namely IL-6, and TNF-α. Taken together, we demonstrated that a novel maple syrup extract exhibited anti-inflammatory, anti-hyperglycemic and anti-lipogenic activities in vitro. The current study adds to the growing body of in vitro and in vivo data supporting the biological effects and potential health benefits of the novel curcumin formulation Longvida® and the natural sweetener, maple syrup.

. These natural products are typically 'whole extracts' constituting complex mixtures of multiple substances found within the plant rather than a single compound alone. This has arisen out of a basic underpinning of the biological effects of botanical extracts, namely, 'the sum of the whole is greater than its parts' due to synergy, additivity and/or complementary effects among multiple constituents.
A growing body of research suggests that plant natural products, both in purified or extract forms, have anti-inflammatory properties and may impart beneficial effects against diseases with an inflammatory component (3)(4)(5)(6).
Dietary intake of bioactive class of compounds 'phenolics' with antioxidant and anti-inflammatory properties represents one mechanism to combat inflammation and promote overall human health. Polyphenols play a significant role in human nutrition and health, as these potent bioactive constituents are the most abundant phytochemicals in our diet (7)(8)(9)(10). Possible efficacy of phenolics including biological extracts such as grape seed extracts, cocoa extracts, etc or pure constituents like resveratrol, curcumin. on inflammation, glucose and lipid homeostasis has been well published in cell culture studies, animal models and some clinical trials (14,(11)(12)(13) It is now well accepted that inflammation and associated pro-inflammatory processes are centrally linked to several chronic human diseases including cancer, diabetes, cardiovascular and neurodegenerative diseases (15)(16)(17).
Regulatory mechanisms that monitor metabolism and immunity overlap with each other at many stages (18). The factors contributing to metabolic diseases like diabetes and obesity are insulin resistance, impaired glucose and lipid metabolism (19). As stated earlier, along with chronic inflammation, altered balance of pro-inflammatory and inflammatory factors are also involved in the pathogenesis of these metabolic diseases. Multiple complex molecular pathways that involve JNK, NFκB, SOCS, AMPK and PPAR family members are responsible for development of hepatic insulin resistance by inflammation (18).
The Nuclear factor kappa B (NFκB) signaling pathway has been well characterized and is considered to be a predominant signaling pathway in inflammation (15,16). The activated form of NFκB has been reported to be involved in chronic inflammatory diseases such as cancer, atherosclerosis, myocardial infarction, diabetes, arthritis, Alzheimer's disease, osteoporosis, and other (20)(21)(22). Thus, agents that can suppress NFκB activation, such as plant natural products can potentially prevent, delay the onset, or treat NFκBlinked diseases (23)(24)(25). Moreover, many well-known antioxidants that are currently being marketed in nutraceutical preparations, such as resveratrol, curcumin, and pomegranate, are thought to elicit anti-inflammatory effects through downregulation of the NFκB signaling pathway in macrophages (26,27). Hence, in manuscript two and three we have demonstrated the antiinflammatory activities of standardized curcumin formulation Longvida® and polyphenolic maple syrup extract in RAW 264.7 macrophage model.
In the first manuscript, 'Anti-inflammatory effects of novel standardized solid lipid curcumin formulations', we have investigated in vitro biological effects of Longvida®, a commercially available standardized novel curcumin formulation, on the NFκB signaling pathway which is a major molecular pathway involved in inflammation (15,16). The investigations carried out in last few decades, confirmed that curcumin (the most active constituent of the curry spice turmeric) targets many transcription factors (NFκB, ATF3, AP-1, STAT-3), inflammatory mediators (NO, PGE 2 , cytokines), enzymes, growth factors, protein kinases and cell-cycle regulatory proteins (27)(28)(29)(30). Despite the promising health benefits of curcumin, water insolubility, low bioavailability and poor absorption are the limiting factors for the use of curcumin as a potential preventive and therapeutic agent (29,31). Lately, very promising results were observed in an in vivo single-dose pharmacokinetic study of standardized novel solid lipid curcumin particle (SLCP) preparation (Longvida®), which reported 65 fold increased bioavailability of the preparation relative to generic curcumin extract and suggests the potential for sustained release dosage form (32). Furthermore an acute and subchronic toxicity study in rats and mice demonstrated the safety of SLCP and the No Observed-Adverse-Effect Level (NOAEL) was determined to be 720 mg/kg bw/day, the highest dose tested (33). Moreover, a recent study demonstrated that a low dose of a Longvida® (80 mg/ day) can produce a variety of potentially health promoting effects in healthy middle-aged people (34 AMPK, a heterotrimeric serine/threonine kinase, is a vital metabolic regulator of fatty acid and glucose homeostasis. It is emerging as a potential target for the treatment of diabetes and chronic inflammatory diseases (19). Numerous studies have reported that the activation of AMPK can inhibit NFκB signaling indirectly, a master regulator of inflammation, both in vitro and in vivo (36).
Recently, activation of AMPK by metformin, a first-line anti-diabetic drug, has been shown to decrease glucose production and increase fatty acid oxidation in the liver (37,38). It has been reported that AMPK activation by polyphenols may be at least partially responsible for their therapeutic benefits on hyperlipidemia in diabetes (39).

Abstract
Inflammation and the presence of pro-inflammatory cytokines are associated with numerous chronic diseases such as type-2 diabetes mellitus, cardiovascular disease, Alzheimer's disease, and cancer. An overwhelming amount of data indicates that curcumin, a polyphenol obtained from the Indian spice turmeric, Curcuma longa, is a potential chemopreventive agent for treating certain cancers and other chronic inflammatory diseases. However, the low bioavailability of curcumin, partly due to its low solubility and stability in the digestive tract, limits its therapeutic applications. Recent studies have demonstrated increased bioavailability and health promoting effects of a novel solid lipid particle formulation of curcumin (Curcumin SLCP, Longvida ® ). The goal of the current study was to evaluate the aqueous solubility and in vitro anti-inflammatory effects of SLCP formulations using lipopolysaccharide (LPS)-stimulated RAW 264.7 cultured murine macrophages. SLCPs treatment significantly decreased nitric oxide (NO) and prostaglandin-E 2 (PGE 2 ) levels at concentrations ranging from 10-50 µg/mL, and reduced Interleukin-6 (IL-6) levels in a concentration-dependent manner. Transient transfection experiments using a nuclear factor-kappa B (NF-κB) reporter construct indicates that SLCPs significantly inhibit the transcriptional activity of NF-κB in macrophages. Taken together, these results show that in RAW 264.7 murine macrophages, SLCPs have improved solubility over unformulated curcumin, and significantly decrease the LPS-induced pro-inflammatory mediators NO, PGE 2 and IL-6 by inhibiting the activation of NF-κB.

Introduction
Inflammation and associated pro-inflammatory processes are centrally linked to several chronic human diseases including cancer, diabetes, obesity, arthritis, cardiovascular and neurodegenerative diseases [1][2][3][4][5][6] . During inflammation, macrophages play a critical role in managing various immunopathological phenomena, including the overproduction of inflammatory markers such as nitric oxide (NO), prostaglandin-E 2 (PGE 2 ), tumor necrotic factor α (TNFα) and cytokines like interleukin-6 (IL-6) and interleukin-1β (IL-1β). A number of inflammatory stimuli, for e.g. lipopolysaccharides (LPS) and pro-inflammatory cytokines, activate immune cells to produce inflammatory mediators, and these are therefore useful targets in the development of novel anti-inflammatory drugs and in the evaluation of the molecular mechanisms of potential anti-inflammatory drugs 7,8 . Thus, dietary agents that can suppress inflammatory markers, such as plant natural products, can potentially prevent, delay the onset, and/or treat inflammation and inflammatory-mediated diseases. A growing body of research suggests that plant phenolic compounds which possess antioxidant and anti-inflammatory properties offer an attractive dietary strategy to combat inflammation and promote human health and wellness 9,10 .
Curcumin (diferuloylmethane) is derived from the ground rhizomes of the Curcuma longa L. plant and is the most active curcuminoid in the Indian curry spice, turmeric 11,12 . Curcumin is a lipophilic, water insoluble, low molecular weight polyphenol (MW = 368 g/moL), which has been used for culinary applications in many parts of the world. In Ayurveda, turmeric is widely used to treat a variety of conditions ranging from acute infections, wounds, and injuries in addition to chronic diseases like diabetes, asthma, and various inflammatory diseases [13][14][15] . A vast number of published research studies support a wide range of pharmacological effects of curcumin including anti-oxidant, anti-cancer, anti-Alzheimer's disease and anti-inflammatory effects both in vitro and in vivo 13,16 . On a molecular level, curcumin targets many transcription factors (including NF-κB, AP-1, STAT-3), inflammatory mediators (PGE 2 , cytokines), enzymes, growth factors, protein kinases and cell-cycle regulatory proteins 13,[17][18][19] Despite the promising health benefits of curcumin, its low water solubility limits its oral delivery in aqueous-based formulations which are popular among consumers and widely used in the nutraceutical and functional food industries.
Moreover, the poor bioavailability and extensive phase-II metabolism of curcumin are limiting factors for the oral dosage of unformulated curcumin which limits its potential as a preventive and/or therapeutic agent 19,20 . Various Clinical trials have reported low systemic bioavailability of curcumin even after oral administration of doses up to 12 g/kg/day 16,21 . Several research strategies have been undertaken to improve the bioavailability of curcumin, including blocking non-specific metabolic pathways, as well as novel drug delivery systems and formulations like liposomes, nanoparticles and phospholipid complexes 13,22 . In a clinical study, a combination of curcumin and piperine, a nonspecific CYP-450 and (UDP-glucuronosyltransferase) UGT inhibitor, resulted in increased curcumin bioavailability, but with an absorption half-life of only 7 minutes 23 . Other in vitro studies suggest that polymer-based nanoparticle formulations of curcumin, also known as 'nano-curcumin', exhibit pharmacological activity at slightly lower concentrations than that of pure curcumin in human pancreatic cancer cell lines 24 . However, the potential increase in bioavailability of these latter formulations is offset by the lack of data and clinical trials on the safety and metabolism of nanoparticle formulations in humans.
Recently, a single-dose human pharmacokinetic study of a standardized novel solid lipid curcumin particle (SLCP) preparation (commercially available as Longvida®) reported increased bioavailability compared to a generic curcumin extract, suggesting the potential for sustained release dosage forms of this natural product 25  (2012) demonstrated that a low dose of a Longvida® (80 mg/day) imparted potentially health promoting effects in healthy middle-aged humans 27 . Also, in a recent investigation, Longvida® selectively suppressed soluble Tau dimers and corrected molecular chaperone, synaptic, and behavioral deficits in transgenic mice suggesting that this curcumin formulation may have potential beneficial effects against Alzheimer's disease 28 . However, to date, the effects of this bioavailable curcumin preparation, namely, Longvida®, on inflammation and inflammatory biomarkers remains unknown.
The goal of the present study was to evaluate the effects of two novel SLCP preparations using LPS-stimulated RAW 264.7 macrophages, a wellestablished in vitro anti-inflammatory model. LPS increases inflammatory markers such as NO, PGE 2 and IL-6 and we hypothesized that the standardized SLCP formulations would block the expression of proinflammatory mediators.

Solid Lipid Curcumin Particle (SLCP) Formulations and Curcumin Extract
The solid lipid curcumin particle (SLCP) preparation (Longvida®, SLCP-1), a solution-dispersible SLCP preparation (Longvida® SD, SLCP-2), and a curcumin extract (containing 95 % curcuminoids) were provided to our laboratory by Verdure Sciences (Noblesville, IN, USA). The SLCP extracts were produced using patent-pending methodology as described previously 25,27 and were standardized to contain approximately 20 % curcumin 25,26 . SLCP-1 is a granular powder used for tablets and capsules, and SLCP-2 is a fine powder intended for use in other dosage forms. Briefly, turmeric root extract was mixed with pure phosphatidylcholine, vegetable stearic acid, ascorbic acid (vitamin C) palmitate, and other inert ingredients. The formulations were manufactured under cGMP standards and meet internal and external specifications for precise chemical and physical characteristics. The solubility of the two SLCP formulations and the curcumin extract are shown in Table 1 (data provided by Verdure Sciences). Cell culture supernatants were collected for nitrite (NO), PGE2, and cytokine assays.

Nitrite determination
RAW 264.7 macrophages were plated in a 96-well plate (1 × 10 5 cells/100 µL) and incubated at 37 °C for 24 h. After 24 h, the medium was replaced and the cells were co-treated with 50 ng/mL LPS and different concentrations of the SLCP formulations (10, 25 and 50 µg/mL) for 24 h at 37 °C. Cells that were not treated with LPS served as a negative control. After 24 h, the cell culture supernatants were collected and incubated (1:1, v/v) with modified Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in distilled water) (Sigma-Aldrich, St. Louis, MO) for 20 min at room temperature as previously described 30 . The absorbance at 540 nm was measured using a spectrophotometer (SpectraMax M2, Molecular Devices Corp, Sunnyvale, CA). The nitrite concentration was quantified by comparison with a sodium nitrite standard curve. The assay was performed in triplicate for each concentration.

PGE 2 determination
To evaluate the effects of various concentrations of the SLCP-1 and SLCP-2 formulations on PGE 2 levels, PGE2 metabolites accumulated in the cell supernatants were measured using a PGE2 enzyme-immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's protocol.
The assay was performed in triplicate for each concentration.

Determination of IL-6
The effects of the SLCP-1 and SLCP-2 formulations on the production of proinflammatory cytokine IL-6 was determined by Bio-Plex® Multiplex Immunoassays (BioRad Laboratories, Hercules, CA), as described by the manufacturer's instructions. The experiment was performed in quadruplicate.

Transient transfection and luciferase assay
LPS-induced NF-κB upregulation accounts for a part of LPS-mediated activation of variety of inflammatory genes and hence it is important to identify the effects of SLCP formulations on the transcriptional activity of NF-κB 31 . To monitor these effects of the two SLCP formulations, RAW 264.7 macrophages were transiently co-transfected with pNF-κB and pRL-CMV reporter vectors (Promega, Madison, CA) using GenePORTER® 3000 Transfection Reagent (Genlantis, San Diego, CA) according to the manufacturer's protocol. Briefly, the cells were seeded into 96-well plates (1.2 × 10 4 cells/100 µL) in complete medium without antibiotics (DMEM +10 % FBS) and incubated for 24 h (~50-70 % confluency). Cells were then treated with the transfection complexes and incubated for 72 h before treatment with each SLCP formulations (10, 25, and 50 µg/mL) for 2 h. LPS was added after 2 h (1 µg/mL). After 5 h, cells were lysed with 20 µL passive lysis buffer and NF-κB activity was measured as relative firefly/renilla luciferase activity using a Dual-Luciferase® Reporter Assay System (Promega, Madison, CA) with a GloMaxTM 20/20 Luminometer (Promega, Madison, CA) according to the manufacturer's protocol. All samples were tested in quadruplicate. Luciferase activity was recorded as relative light units (RLU) and expressed as fold change relative to LPS-treatment control and the assay was performed using three replicates for each concentration.

Statistical analysis
All statistical analyses were carried out using the software program GraphPad Prism Version 5.0 (GraphPad Software, La Jolla, CA). Experimental data were grouped by one variable and were analyzed by one-way ANOVA followed by a Dunnett's multiple comparison test. A value of p < 0.05 was considered significant.

Aqueous solubility of SLCP formulations vs. curcumin
The SLCP-1 and SLCP-2 formulations were both soluble in water, with SLCP-1 possessing ca. 14 % solubility and SLCP-2 having ca. 76 % solubility (see Table 1). On the other hand, the curcumin extract exhibited significantly less solubility in water which was in agreement with the previous data published by Kurien et al. (2007) 32 . Given this solubility data as well as the previously published data supporting the increased bioavailability of the SLCPs compared to a generic curcumin extract in humans, 25 we proceeded to further evaluate the SLCP formulations in targeted in vitro bioassays (described below).

Effect of SLCP formulations on LPS-induced NO production
NO and PGE 2 are secreted into cell culture supernatant by RAW 264.7 murine macrophages when they are treated with LPS. Since NO is highly unstable, the accumulation of nitrite (a stable oxidized product of NO) in culture media is often used as a biomarker for NO production in LPS-activated macrophages 33 .
A nitric oxide assay was used to evaluate the effect of the SLCP formulations on NO production as described in section 2.4. RAW 264.7 cells were incubated with or without LPS (50 ng/mL) in the presence or absence of test samples (10,25 and 50 µg/mL) for 24 h. In LPS-stimulated macrophages, nitrite levels increased significantly to 88.7 ± 6.2 µM as compared to the solvent control (Fig. 1A). In Fig. 1A, treatment with SLCP formulations suppressed nitrite concentration in LPS-activated macrophages in a concentration-dependent manner. At 25 µg/mL, both SLCP formulations significantly decreased medium nitrite to 33.5 ± 4.2 µM and 61.6 ± 2.0 µM respectively. At 50 µg/mL, SLCP-1 and SLCP-2 both inhibited LPS-induced NO production in macrophages by 100 %. No nitrite production could be detected in cells treated with test compounds without LPS (data not shown).

Effect of SLCP on PGE 2 production
We next evaluated the effects of SLCP on LPS-induced PGE 2 synthesis in RAW 264.7 cells. The same supernatants were used for measurement of PGE 2 . After stimulation with LPS (50 ng/mL), PGE 2 was released into the culture medium and rapidly converted into its metabolite. As shown in Fig. 1B, at 10 µg/mL SLCP-1and SLCP-2 lowered LPS-induced PGE 2 production to about 31 and 25 %, respectively. Treatment with 25 µg/mL of SLCP-1 and SLCP-2 significantly suppressed PGE 2 levels by about 66 and 64 % respectively, whereas both the test compounds at 50 µg/mL concentration reduced PGE 2 levels to about 96 % as compared to control. The SLCP formulations did not significantly increase PGE 2 levels in cells which were not treated with LPS (data not shown). Thus our results show that SLCPs inhibited PGE 2 production in a concentration-dependent manner in LPS-stimulated cells. The test compounds were not responsible for altering the viability of activated macrophages as determined by MTS assay (see Supplementary   Fig.1). Hence, the observed reduction in NO and PGE 2 production by the SLCP treatments was not attributed to cytotoxic effects.

Effects of SLCPs on the levels of IL-6
LPS induction in macrophages causes the up-regulation of pro-inflammatory cytokines such as IL-6 34 . Consequently, the effect of varying concentrations of the test compounds on this cytokine was evaluated. LPS treatment significantly elevated levels of IL-6 (6043 ± 589.94) in the media (Fig. 2). IL-6 levels in macrophages treated with the test compounds alone, without LPS, were negligible (data not shown). The SLCP formulations suppressed IL-6 levels in a concentration-dependent manner. At 25 and 50 µg/mL of SLCP-1 treatment, IL-6 levels were significantly reduced by 29.4 and 97.4 % respectively. Similarly, treatment with 10, 25 and 50 µg/mL of SLCP-2 decreased IL-6 production by about 2, 13 and 95 %, respectively. This data suggested that, both SLCP formulations were effective in lowering IL-6 levels in LPS-stimulated macrophages, with SLCP-1 being slightly more effective than SLCP-2 at higher concentrations.

Effects of SLCP formulations on NF-κB activation
The activation of the transcription factor, NF-κB is a key-step in stimulating pro-inflammatory signals. Thus, we next investigated the effect of the SLCP formulations on NF-κB activity, by transient transfection of macrophages followed by luciferase assay. After treatment with 1 µg/mL LPS, the NF-κB activity of the macrophages was significantly increased as compared to control (Fig. 3). Treatment with the SLCPs formulations significantly reduced NF-κB transcription activity in a concentration dependent manner, as shown in Fig. 3.
NF-κB luciferase activity was decreased to almost 10-fold with 10 µg/mL of SLCP-1 treatment, whereas with 10 µg/mL of SLCP-2, the activity was reduced to about 6-fold as compared to control.

Discussion
Inflammation is a key component in multiple disease states 35 . Based on a review of the literature, the NF-κB signaling pathway is the predominant upstream molecular signaling pathway that causes inflammation through enhanced cytokine, nitric oxide, and prostaglandin production 36 . Several studies in different animal models and in human trials support the diverse pharmacological effects of curcumin including anti-proliferative, antiangiogenic, anti-oxidant, anti-inflammatory, anti-microbial, hepato-and nephro-protective properties etc. 22 . The SLCPs are novel proprietary curcumin formulations with improved solubility and bioavailability compared to generic curcumin. 25 In this study, we investigated the inhibitory effects of the SLCP treatments on the inflammatory response initiated by LPS in RAW 264.7 macrophages.
In inflammation, macrophages undergo sequential steps to release proinflammatory mediators like cytokines, NO and PGE 2 . These molecules recruit other immune cells to the sites of inflammation. Therefore inhibition of the release of these chemicals is a good strategy for monitoring inflammatory diseases 35 .
In the current study, we reported that LPS-activated RAW 264.7 murine cells treated with improved-solubility curcumin formulations, namely, SLCP-1 and SLCP-2, exhibited concentration-dependent down regulation of NO and PGE 2 production. These observations were in agreement with the previous studies, which reported the inhibition of NO through the suppression of inducible, NO synthase gene and protein expression by curcumin in LPS and IFN-γ activated RAW 264.7 macrophages 37,38 . Our study further revealed that the SLCP treatments on LPS-activated macrophages decreased IL-6 levels in a concentration dependent manner. It has been demonstrated previously that there is a reduction in LPS-induced IL-6 levels with curcumin treatment in macrophages 39,40 . Moreover, it was reported that curcumin blocked the LPSinduced NF-κB activation through the prevention of Inhibitor κB degradation in RAW 264.7 cells 38 . Thus, the down regulation of NF-κB activation can be one of the many mechanisms underlying the anti-inflammatory effects of the SLCPs.
The improved solubility of SLCP formulations compared to regular curcumin (see Table 1) may be due to the amphiphilic nature of SLP formulations that utilize phospholipids and other lipids to solubilize curcumin in aqueous solutions. In previous studies, similar types of formulations contributed to the creation of micelle type physical structures that permit increased dissolution of lipophilic compounds. SLCP-1 and SLCP-2 differ with respect to powder size, and the differential solubility between them (Table 1) may be due to the relative amount of surface area exposed to the aqueous medium. In this study, it was found that SLCP-2 possessed greater water solubility than SLCP-1, likely due to its decreased powder size and thus increased surface area exposed to the aqueous solution. However, both SLCPs possessed similar activity in vitro on inflammatory mediators. Future research may investigate the dissolution and micellar characteristics of these formulations and their interactions with cytokines on a molecular level. Overall, the increased aqueous solubility of the SLCP formulations compared to natural curcumin leads to a broader scope of possible formulations which can be utilized by the nutraceutical and functional food industries.
In toto, solid lipid curcumin particle (SLCP) formulations dosedependently mitigated the LPS-induced inflammatory response in macrophages, by down regulation of the production of the inflammatory markers NO, PGE 2 and IL-6 through inhibition of NF-κB activation. SLCP-1 was slightly more effective at inhibiting the anti-inflammatory response than SLCP-2, and this may be due to concentration-dependent effects. The current findings suggest the use of increased-water soluble curcumin formulations such as SLCPs as potential therapeutic agents to combat chronic inflammatory diseases. Further investigations should explore the potential use of SLCP in the prevention and/or therapy of inflammation-linked diseases but future in vivo studies on SLCP are warranted to determine the clinical efficacy of these formulations of curcumin.

Acknowledgments
This project was partially supported by Verdure Sciences (Noblesville, IN) who also kindly provided the SLCP formulations and curcumin extract. This work was also supported by grants from the National Institute of Health

Author Disclosure Statement
The authors declare no competing financial interests.

Introduction
Functional foods and nutraceuticals, consumed for human health promotion and disease risk reduction, are among the fastest growing sectors of the modern food industry (Hardy, 2000). This has led to increased interest into the discovery and applications of new functional ingredients, from both marine and terrestrial sources, by several research laboratories (Marete, Jacquier, & O'Riordan, 2011;Vo & Kim, 2013). Similarly, our group has been involved in the identification of bioactive compounds from maple (Acer) species and maple syrup , 2011a, 2011b. Notably, our research efforts in this area have contributed, in part, to the recent commercial development and utilization of maple sap/water as a functional beverage (Yuan, Li, Zhang & Seeram, 2013; http://ilovemaple.ca/products/maple-water).
In the current project, we have now focused our attention on investigating the biological activities of a 'phenolic-enriched and sugar-reduced' extract derived from maple syrup (further explained below) for future potential applications as a functional food ingredient.
Maple syrup is a natural sweetener produced by concentrating the watery sap collected from maple species including the sugar maple (Acer saccharum) and red maple (Acer rubrum) species, which are both native to North America Perkins & van den Berg, 2009). Maple syrup contains sugars (mainly as sucrose), organic acids, amino acids, minerals, and phytochemicals, the latter of which occur primarily as phenolics (Abou-Zaid, Nozzolillo, Tonon, Coppens, & Lombardo, 2008;Ball, 2007;. As mentioned above, our laboratory has previously isolated and identified more than fifty phenolics from maple syrup belonging to the lignan, coumarin, stilbene, and phenolic derivative sub-classes , 2011a, 2011b. However, until now, the anti-inflammatory activity and the associated molecular mechanisms elicited by phenolic-enriched maple syrup extract have not been thoroughly studied. Furthermore, the activities of the purified phenolic compounds isolated from maple syrup that may be responsible for any observable anti-inflammatory activities have not been reported.
Inflammation is implicated in multiple chronic human diseases, such as cancer, diabetes, cardiovascular and neurodegenerative diseases.

Moreover, previous research has shown that plant extracts and their purified
compounds inhibit the pro-inflammatory COX-2 enzyme and the transcription factor, NF-κB Rettig, et al., 2008).
In the present study, we examined the potential for a phenolic-  Table 1), previously isolated from maple syrup by our group , 2011b, were evaluated for their effects on NO and PGE 2 production in the LPS-stimulated macrophages.

Phenolic-enriched maple syrup ethyl acetate extract (MS-EtOAc)
Maple syrup was donated to our laboratory by the Federation of Maple Maple

Isolation and identification of pure compounds from maple syrup
The isolation and identification of compounds from maple syrup have been previously reported , 2011b. The maple syrup isolates are predominantly phenolics belonging to lignan, coumarin, stilbene, and phenolic derivative sub-classes. Unfortunately, since many of the compounds were isolated in small quantities, and we were limited by sample availability, we were only able to pursue studies on 15 of the pure compounds (identities are shown in Table 1; chemical structures are shown in Fig. 1). In addition, because of limited quantity of these pure samples, we were only able to evaluate each sample once (in a 96-well format) but at three different concentrations for the bioassays described below. was used as a positive control for the assays, as it has been previously described to decrease LPS-induced NO and PGE 2 induction in RAW 264.7 cells through NF-κB downregulation (Djoko, et al., 2007;Tsai, Lin-Shiau, & Lin, 1999). After 24 h incubation, cell culture supernatants were collected for NO and PGE 2 measurements. Cell lysates were collected for both gene expression and protein expression analyses. For gene expression studies, RAW 264.7 cells were seeded in a 24-well plate at 1 × 10 6 cells/500 µL density. For Western blot analysis, cells were plated in a 6-well plate at 2 × 10 6 cells/mL density. Cell supernatants and lysates were stored at -80°C until use.

Cytotoxicity Assay
The viability of RAW 264.7 macrophages after 24 h of continuous exposure to MS-EtOAc extract (10-100 µg/mL) or the pure compounds (

Nitric oxide (NO) activity
The nitrite levels in the cell culture supernatants were assayed as an indicator of NO production, according to the Griess reaction as previously described by for 20 min at room temperature. The optical densities were measured at 540 nm and NO concentration was determined by comparison to a standard curve.
The assay was performed in triplicate and repeated at least 4 times.

Prostaglandin E 2 release
The Prostaglandin E 2 Metabolite (PGE 2 ) assay is based on the conversion of all major PGE 2 metabolites into a single stable derivative, which is measured by using an enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). Briefly, the protocol was followed using the manufacturer's protocol:

Western blotting
To extract protein for Western blot analyses, cells were harvested and washed with PBS. The whole cell protein extracts were prepared using RIPA buffer. min. Bands were visualized on X-ray films using ECL chemiluminescence detection kit (GE Healthcare, Piscataway, NJ) according to the manufacturer's manual.

Transient transfection and luciferase assay
LPS-induced NF-κB upregulation accounts for a part of LPS-mediated activation of variety of other inflammatory genes. Hence it is important to identify the effects of MS-EtOAc extract on the transcriptional activity of NF-κB (Djoko, et al., 2007). Luciferase activity was recorded as relative light units (RLU) and expressed as fold change relative to LPS-treatment control and the assay was performed using three biological replicates.

Statistical analysis
All statistical analyses were carried out using the software program GraphPad Prism Version 5.0 (GraphPad Software, La Jolla, CA). Experimental data were grouped by one variable and were analyzed by unpaired two-tailed t-test or one-way ANOVA followed by a Dunnett's multiple comparison test. A value of p < 0.05 was considered significant.

MS-EtOAc extract inhibit NO production in LPS-stimulated RAW 264.7 cells
In order to study the anti-inflammatory activity of maple syrup phenolicenriched extract, a well-known inflammatory marker, namely NO, was selected. The effects of MS-EtOAc extract on NO production was determined by the measurement of nitrite released into the culture supernatants by LPS stimulated RAW 264.7 cells using the Griess reagent (Guevara, et al., 1998;Legault, et al., 2010). Nitrite levels in unstimulated RAW 264.7 cells were at baseline levels (2.8 ± 0.2 µM). However, upon stimulation with LPS, nitrite accumulation in the culture supernatant increased ~22-fold to 60.83 ± 2.2 µM.
Resveratrol, a known inhibitor of NO activity (Djoko, et al., 2007;Wadsworth & Koop, 1999), was used as a positive control for comparing the activity of MS-EtOAc. Resveratrol (10 µg/mL) inhibited NO release by 88.67% in LPSstimulated RAW 264.7 macrophages. Similar activity was obtained with MS-EtOAc (Fig. 2a), which significantly reduced nitrite production by 84.9% (at 50 µg/mL) and 94.3% (at 100 µg/mL) as compared to the nitrite production by

MS-EtOAc inhibits PGE 2 production in LPS-stimulated RAW 264.7 cells
PGE 2 also functions as a mediator of inflammation, which is produced by metabolism of arachidonic acid by COX enzymes at inflammatory sites (Posadas, et al., 2000). The inhibitory effects of MS-EtOAc extract on PGE 2 levels in LPS-stimulated macrophages were evaluated. As shown in Fig. 2b Fig. 1).
Hence, the observed reduction in NO and PGE 2 production by the MS-EtOAc treatment was not attributed to cytotoxic effects.

Pure maple syrup isolates inhibit NO and PGE 2 production in LPSstimulated RAW 264.7 cells
Because MS-EtOAc is a complex mixture of compounds that could account for its NO and PGE 2 inhibitory effects, we tested 15 of its pure isolates for their ability to reduce NO and PGE 2 production. These compounds were previously isolated from maple syrup and their structures were elucidated by NMR methods , 2011b. In the MS-EtOAc phenolicenriched extract, compound 1 is the only stilbene, whereas compound 5 is the only coumarin among the tested samples. Compounds 9,10,12,14 and 15 were all lignans. All of the other compounds were phenolic derivatives and are briefly categorized as follows: compound 13 contained a C 6 unit; compound 11 contained a C 6 -C 1 unit; compounds 2, 3 and 7 contained a C 6 -C 2 unit and compounds 4, 6 and 8 contained a C 6 -C 3 unit. At the test concentrations, the pure compounds did not exhibit any cytotoxicity in the RAW 264.7 cells (data not shown).
Among all of the pure isolates, the only stilbene, (E)-3,3'-dimethoxy-4,4'-dihydroxystilbene (compound 1), was the most active compound and significantly inhibited NO as well as PGE 2 production in a concentration dependent manner (Table 1). At 50 µM, compound 1 decreased the nitrite level by 92.5% and PGE 2 level by 89.5% in media. These results were similar to the effects of the well-known stilbene, resveratrol (50 µM; used as a positive control), on the above inflammatory markers (data not shown).
As shown in Table 1, apart from compound 1, the majority of the other pure compounds were not effective in modulating NO production in LPSstimulated macrophages. However, compounds 3, 7, and 11 reduced PGE 2 levels in a concentration-dependent manner. At 50 µM concentrations, compounds 3, 7 and 11 were the most effective in lowering PGE 2 levels with 95, 72.6, and 55.3% decrease, respectively. Interestingly, in this assay, the two most active isolates, compounds 3 and 7, both contained a C 6 -C 2 structural motif. However, compound 2, which also contained the same C 6 -C 2 structural motif, did not show any activity against the inflammatory markers.
Based on structure activity related observations, the active compound 3 contained two aromatic hydroxyl groups while the inactive compound 2 contained three aromatic hydroxyls. Moreover, the active compound 7 only contained one aromatic hydroxyl group. Therefore, it would appear that ≤ two aromatic hydroxyls are required for inhibition of PGE 2 levels but further studies on a larger number of structurally diverse compounds would be required to confirm this. Interestingly, compound 7, protocatechuic acid, has been shown to have anti-oxidant, anti-inflammatory and anti-cancer activity in vitro and in vivo (Lende, et al., 2011;Nakamura, et al., 2000;Reis, et al., 2010;Robbins, 2003).
Our data with compound 11, tyrosol, a well-known constituent of olive oil, was in agreement with a previous study where the reported IC 50 value for tyrosol for lowering NO level was >100 µM (Choe, et al., 2012). Tyrosol (at 1, 2 and 4 mM concentrations) has been shown to inhibit iNOS and COX-2 expression in RAW 264.7 cells stimulated with IFN-γ and gliadin through reduction in NF-κB, interferon regulatory factor-1 (IRF-1) and signal transducer and activator of transcription-1α (STAT-1α) activity (De Stefano, et al., 2007).
However, this is the first report to show that tyrosol (at 50 µM test concentration) inhibits PGE 2 level in RAW 264.7 cells stimulated by LPS.
Based on the above observations (detailed in Sec. 3. 1 -3.3), our findings suggest that the unique combination of phenolic phytochemicals in MS-EtOAc extract may be responsible for its anti-inflammatory activity. It is possible that these compounds work complementarily, additively, and/or synergistically in toto to abate inflammatory processes. Unfortunately, several of the previously reported pure compounds from MS-EtOAc were not isolated in sufficient quantities to facilitate further biological assaying , 2011b. Thus, future studies to evaluate the effects of all of the purified phenolic compounds from MS-EtOAc are warranted.
During transformation of maple sap into syrup, unique phenolic and non-phenolic compounds are formed due to the intensive heating process. For example, Quebecol, a novel process derived phenolic compound has been reported in maple syrup (Li & Seeram, 2011a). It has been demonstrated that transformation of maple sap in syrup improves NO inhibition activity. The heating process involved in the transformation of maple sap in syrup induces oxidation of the phenolics, suggesting its implication in the activity . Further research is needed to identify these compounds as they may contribute to the observed biological effects of maple syrup.

Effect of MS-EtOAc extract on iNOS and COX-2 mRNA and protein expression in LPS-stimulated RAW 264.7 murine macrophages
In macrophages exposed to LPS, iNOS and COX-2 mRNA and protein expression are modulated, which ultimately results in overproduction of NO and PGE 2 . In order to assess whether the inhibitory effects of MS-EtOAc extract on NO and PGE 2 were related to the alteration of iNOS and COX-2 enzymes, we examined their relative gene and protein expression by qRT-PCR and western blot analysis, respectively. iNOS and COX-2 mRNA and proteins were barely detectable in un-stimulated cells but were strongly expressed in lysates of LPS-stimulated RAW 264.7 cells. Resveratrol significantly decreased iNOS mRNA and protein levels at 10 µg/mL, which is in agreement with the previous reports wherein gene activation of iNOS was inhibited by resveratrol (Tsai, et al., 1999). MS-EtOAc decreased iNOS mRNA expression in a concentration-dependent manner, by 51% and 72%, at 50 and 100 µg/mL, concentrations, respectively (Fig. 3a). Not surprisingly, MS-EtOAc extract also inhibited iNOS protein expression in a concentration-dependent manner (Fig. 3b). These results suggest that MS-EtOAc extract likely decreases nitrite accumulation through decreased iNOS gene and protein expression.
The acquired data clearly indicates that MS-EtOAc extract did not inhibit COX-2 mRNA (Fig. 4a) and protein levels ( Fig. 4b) but inhibited PGE2 production as discussed previously. Since COX-2 gene expression was upregulated by MS-EtOAc extract treatment, the inhibitory effects observed in regards to decreased PGE 2 levels may potentially occur at the translational or post-translational levels. The reduced PGE 2 levels could be due to the inhibition at the enzymatic level by MS-EtOAc treatment. Resveratrol has also been shown to have a direct inhibitory effect on PG production by suppressing COX-1 and COX-2 enzyme activity in a concentration-dependent manner (Jang, et al., 1997;Subbaramaiah, et al., 1998).
MS-EtOAc demonstrated a similar mechanism of action common to non-steroidal anti-inflammatory drugs (NSAIDs) in inhibiting PGE2 production.
There is a complex 'cross-talk' between the iNOS and COX-2 pathways as has been evidenced by numerous studies. There are reports suggesting that NO seems to decrease COX-2 expression, however, not all published reports are consistent. Inhibitors of nitric oxide synthase activity such as N Gmonomethyl-L-arginine (L-NMMA) have been demonstrated to increase COX-2 protein expression with decrease in NO production in rodent macrophages (Habib, et al., 1997;Patel, Attur, Dave, Abramson, & Amin, 1999;Swierkosz, Mitchell, Warner, Botting, & Vane, 1995). Reduced NO levels may contribute to the observed increased COX-2 protein and mRNA expression with MS-EtOAc treatment. Besides this negative regulation of COX-2 expression, other potential modes of action could be an effect of MS-EtOAc on COX-2 mRNA stability or effects at the transcriptional level. Therefore, further investigation is required to explore how MS-EtOAc affects the COX pathway and whether it inhibits COX activity at the enzymatic level.

MS-EtOAc extract decreases NF-κB response element-luciferase reporter construct activity
Inflammation is associated with high levels of pro-inflammatory mediators like NO and PGE 2, which are produced by the inducible isoforms of enzymes iNOS and COX-2, respectively (Chung, et al., 2008;Nathan, 1992).
The transcription factor NF-κB, which regulates the expression of iNOS and PGE-2, plays a crucial role in inflammatory and immune responses (Chen, Castranova, Shi, & Demers, 1999). Thus, modulating NF-κB activating signals is one of the widely used strategies for the treatment of diseases associated with inflammation. Hence in order to measure NF-κB transcriptional activity, the RAW 264.7 cells were transiently transfected with pNF-κB-luc and pRL-CMV-renilla and reporter constructs were used to determine the effects of MS-EtOAc extract on cells challenged with LPS. As expected, LPS treatment increased activity of the NF-κB promoter construct by about 20-fold as compared to control (Fig. 5). The NF-κB activity was decreased by approximately 50% with 10 and 50 µg/mL concentrations of MS-EtOAc.
Moreover, 100 µg/mL of MS-EtOAc decreased NF-κB reporter construct activity by ~ 75% (Fig. 5). The data herein supports the hypothesis that MS-EtOAc extract may interfere with NF-κB transcriptional activity. LPS-induced iNOS gene activation has been linked to LPS-mediated NF-κB activation (Lowenstein, et al., 1993). Thus downregulation of iNOS expression by MS-EtOAc may be through reduced NF-κB transcriptional activity.
However, inflammation involves multiple complex pathways and these pathways interfere with each other. Thus, NF-κB is not the only transcription factor in LPS-mediated COX-2 gene expression in the mouse macrophages. It has been reported previously that NF-κB site is not essential for LPS-mediated COX-2 gene expression in RAW 264.7 macrophages (Wadleigh, Reddy, Kopp, Ghosh, & Herschman, 2000). This might explain the elevated COX-2 gene and protein expression.
On the basis of current results, we propose that the anti-inflammatory effect of maple syrup extract could be attributed to the inhibition of inflammatory modulators NO and PGE 2 through suppression of NF-κB transcription.

Conclusion
The current study is the first report to investigate the mechanisms underlying the anti-inflammatory activity of a phenolic-enriched maple syrup extract and several of its purified constituents. We found that the maple syrup extract and its purified compounds significantly inhibited the production of LPS-stimulated inflammatory markers NO, PGE 2 and iNOS through the direct inhibition of NF-κB transcriptional activity in RAW 264.7 macrophages. However, the upregulation of COX-2 gene and protein levels remain unexplained. Overall, the cellular mechanisms underlying these multiple effects may be attributed to the unique combination of pure compounds present in the extract, which appear to act in toto. More detailed research is needed to get further insights into the exact anti-inflammatory mechanism of the MS-EtOAc extract. between groups were determined using a one-way ANOVA test followed by a Dunnett's multiple comparison test. # P < 0.05 solvent control compared with the LPS-treated cells; significant difference was determined using unpaired student-t test. concentration dependent manner. ***P < 0.001 as compared with the LPStreated group; significant differences between groups were determined using a one-way ANOVA test followed by Dunnett's multiple comparison test. # P < 0.05 solvent control compared with the LPS-treated cells; significant difference was determined using unpaired student-t test. protein expression. ***P < 0.001 as compared with the LPS-treated group;

Figure legends
significant differences between groups were determined using a one-way ANOVA test followed by Dunnett's multiple comparison test. # P < 0.05 solvent control compared with the LPS-treated cells; significant difference was determined using unpaired student-t test.

Fig. 5 Effects of MS-EtOAc on LPS induced NF-κB luciferase activity in macrophages.
RAW 264.7 cells were transiently co-transfected with pNF-κB and pRL-CMV reporter vectors. NF-κB activity was measured using a Promega dual luciferase assay system. MS-EtOAc significantly reduced the LPS-induced increase in NF-κB-dependent luciferase enzyme expression in a concentration-dependent manner. Data is the mean ± S.D. of three different samples. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with the LPStreated group; significant differences between groups were determined using a one-way ANOVA test followed by Dunnett's multiple comparison test. # P < 0.05 solvent control compared with the LPS-treated cells; significant difference was determined using unpaired student-t test.

Introduction :
In last few decades, the prevalence of type 2 diabetes mellitus (T2DM) has radically increased. Insulin resistance (IR) along with hyperglycemia and hyperinsulinaemia is the hallmark of T2DM. In T2DM, hyperglycaemia is predominantly caused by atypical increase in hepatic gluconeogenesis, leading to drastic increase in glucose levels (1,2). Metformin, a well-known anti-diabetic drug, primarily corrects hyperglycaemia and hyperinsulinaemia by lowering hepatic gluconeogenesis via 5' adenosine monophosphate-activated kinase (AMPK) activation (3).
There is also an established link between obesity and diabetes due to related inflammatory processes (4). Obesity leads to increased circulation of proinflammatory markers leading to 'metabolic inflammation' which has been linked to the pathogenesis of IR and T2DM in humans and animal models (4).
Moreover, maple syrup extracts have been shown to have antioxidant (6), αglucosidase enzyme inhibitory (17), anticancer (18) and anti-inflammatory (19) properties in vitro. In addition, maple syrup has been shown to have liver protective effects (20), improve metabolic response (21), and ability to reduce plasma glucose level compared to a sucrose solution alone (22,23) in animal models. Recently, St-Pierre et al., (2014) reported that maple syrup produced low glycaemic and insulinaemic responses in vivo, and thus represents a natural sweetener alternative to refined sugar (21). Recently, our lab has reported MSX treatment lowered media glucose levels as compared to control in human HepG2 cells (15). However, the mechanism behind hypoglycemic effect of MSX is still unknown. Therefore, given all of above published in vitro and animal data, we believe that further investigation into demonstrating the hypoglycemic and hypolipidemic effects of this novel MSX extract is warranted.
In this investigation, we hypothesized that nutraceutical grade MSX extract lowered glucose levels via AMPK phosphorylation in hepatocytes. Moreover, we postulate the de-lipidating effects of MSX extract by downregulating the lipogenic targets in mature adipocytes.
previously described the preparation and chemical composition of the maple syrup nutraceutical extract (named, MSX), which was used in this study (15).

Glucose Consumption Assay
The glucose concentration in the HepG2 cell supernatants was determined using a glucose assay kit as per the manufacturer's instructions. Absorbance was measured at 490 nm using a spectrophotometer (SpectraMax M2, Molecular Devices Corp, Sunnyvale, CA, USA), and the assay was performed in triplicates.

Oil red O staining and quantification
During differentiation of pre-adipocytes into adipocytes, intracellular lipid droplets are accumulated. ORO dye is used for staining these lipid droplets (26,27

Western blot
The cells were washed once with phosphate-buffered saline and lysed in RIPA buffer. Protein concentration was measured using a bichonic protein assay kit.
Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After 1 h incubation in blocking solution (5% skim milk), the membranes were incubated overnight with primary antibodies in cold room. The membranes were washed thrice with Tris buffered saline/Tween-20 (TBST) for 10 min each followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were again washed three times with TBST and then developed on X-ray films using ECL™ detection reagent. The bands were quantified using the Image J program (NIH, Washington, DC, USA).

Statistical analyses
All statistical analyses were carried out using the software program GraphPad Prism Version 5.0 (GraphPad Software, La Jolla, CA, USA). Results are expressed as the means ± the standard error of the mean (S.E.). The experimental data was analyzed by unpaired one-tailed t-test (for compairing differences between two individual groups) or one-way ANOVA (for comparing differences between all groups together) followed by a Dunnett's multiple comparison test. p < 0.05 was considered significant.

MSX increase glucose consumption in HepG2 cells by downregulating gluconeogenic genes via AMPK activation
It has been reported that natural products can regulate glucose and lipid metabolism in HepG2 liver hepatocytes (28). Similarly, in our recent MSX study (15)  However, PEPCK mRNA expression was similar to vehicle-treated controls ( Fig. 2).
In order to conduct further investigation into gluconeogenic pathway, upstream regulator AMPK protein expression was studied. AMPK activation is commonly detected by monitoring its phosphorylation at Thr-172 and phosphorylation of downstream target acetyl-CoA carboxylase-1 (ACC-1), using phospho-specific antibodies. ACC-1 is a key enzyme involved in fatty acid synthesis, which is phosphorylated and inactivated by AMPK (32). MSX (100 µg/mL) slightly increased the ratio of pAMPK/ AMPK and pACC-1/ ACC-1 by about 40% and 26% of control, respectively (Fig. 3).
Moreover, MSX reduced the expression of PEPCK protein to about 55% and 43% of control at 50 µg/mL and 100 µg/mL concentration, respectively. Thus, MSX could be inhibiting PEPCK at translational level ( Fig. 3) but not transcriptionally (Fig. 2).
Thus, glucose-lowering effect of MSX at higher concentration (100 µg/mL) could be attributed to downregulation of G6pase gene expression and PEPCK protein expression through AMPK activation. These results supported the published in vivo studies with maple syrup; which reported reduced plasma glucose levels compared to a sucrose solution alone in an anti-diabetic animal model (22,23). However, this is a preliminary study and further more detailed in vivo studies are needed to support this data.  (24,25,33). Resveratrol, at the concentration selected, reduced the staining to 78% of vehicle-treated control adipocytes (Fig. 4B). In contrast, metformin and MSX (50 and 100 µg/mL) decreased the ORO staining by approximately 40-50% of control (Fig. 4B). To put our data in context with human health perspective, we tested MSX in mature adipocytes from a human donor. Metformin and MSX treatment reduced ORO staining in human adipocytes to 64% and 43% of vehicle-treated controls, respectively (Fig. 4D).

MSX downregulated lipogenic targets in mature 3T3-L1 adipocytes.
In metabolic syndrome, adipocytes play crucial role in lipid storage and metabolism (34). In adipocytes, several transcription factors act in concert during the adipogenesis and lipogensis phase, including PPARγ, C/EBPα, and SREBP-1 (34)(35)(36). In order to explore the molecular mechanisms involved in MSX anti-lipogenic effects, the cell lysates were collected after 48 h treatment for evaluating lipogenic target protein expression in mature 3T3-L1 adipocytes. It is now well accepted that inflammation and associated pro-inflammatory processes are centrally linked to several chronic human diseases including obesity and diabetes (40). Hence, after exhibiting anti-lipogenic effect, MSX also demonstrated specific effects on mature adipocytes that likely contribute to its overall anti-inflammatory effects. TNF-α and IL-6 are two key mediators of the inflammatory response in mature 3T3-L1 adipocytes that have been linked to obesity-related inflammation. Thus, MSX effect on adipocyte inflammation was explored by measuring TNF-α and IL-6 mRNA expression ( Fig. 6). At 48 hours, resveratrol reduced TNF-α or IL-6 mRNA expression to 60% and 40% of control respectively. In addition, MSX treatment at 50 µg/mL also reduced TNF-α and IL-6 to about 20% of control, however 100 µg/mL MSX treatment reduced both inflammatory targets to 36-47%. This is in agreement with our previous research where we have exhibited antiinflammatory effects of MSX (15) and maple syrup ethyl acetate extract (16) in lipopolysaccharide-induced macrophages. Moreover, in this study we have demonstrated inhibitory effect of MSX treatment on inflammatory markers in mature adipocytes. This is not surprising as MSX contains high levels of polyphenols, which are well-known antioxidants and anti-inflammatory agents (41). Interestingly, MSX contains stilbenes which are structurally related to resveratrol, a well-known polyphenol, thus we could anticipate MSX antilipogenic and anti-inflammatory effects (15).
MSX also contains high quantity of phytohormone abscisic acid (ABA), which has been proposed to improve insulin sensitivity and obesity-related inflammatory diseases through a PPAR γ-dependent mechanism. ABA is structurally similar to thiazolidinediones (drugs prescribed for diabetic management) (42). Dietary abscisic acid has been reported to improve glucose tolerance and obesity-related inflammation in db/db mice fed high-fat diet (43). MSX is particularly rich in lignans and these polyphenolic compounds are also found in flaxseed. Interestingly, flaxseed lignan have been shown to inhibit fat accumulation and induce adiponectin expression in high-fat diet-induced mouse model (44).
In conclusion, as depicted in Fig. 7 the lowered glucose levels after MSX treatment were attributed to AMPK activation in HepG2 hepatoma cell model.
However, the effect of MSX-treatment were not drastic in HepG2 cells.
Moreover, we demonstrated delipidating effects of MSX extract treatment by downregulating protein expression of adipo/lipogenic transcription factors and their downstream targets in differentiated 3T3L1 adipocytes (Fig. 6). In future, it would be interesting to explore mechanistic insights into obesity-linked inflammatory pathway using MSX-treated human adipose tissue explants.
This study, using in vitro models, provided preliminary data necessary to guide and support future in vivo studies investigating the potential use of a novel food grade maple syrup extract (MSX) as a nutraceutical preparation for positive impact on overall health.       5. In conclusion, this study, using in vitro models, provided preliminary data necessary to guide and support future in vivo studies investigating the potential use of a novel curcumin formulation Longvida® and food grade maple syrup extract (MSX) as a nutraceutical preparation for positive impact on overall health.