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Suppression of Inflammatory Mediators by Ethanol Extracts of Pennisetum purpureum S. (Napiergrass Taishigrass no. 2) in Activated RAW 264.7 Macrophages

Yue-Hwa Chen, Yi-Han Wang, Yun-Ting Chen, Yuan-Yu Hou, Sassy Bhawamai, Yu-Wei Fang*

Division of Nephrology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan, ROC

Abstract: Napiergrass Taishigrass no. 2 (NT2) is a new hybrid of napiergrass (Pennisetum purpureum S.), and is commonly added as an ingredient in bioorganic energy soup, a plant-based drink which is thought to have health-promoting effects. Thus, NT2 is hypothesized to possess biological functions. In this study, we explored the effects of NT2 ethanol extracts on in vitro antioxidant activities and on modulation of immune mediators in RAW 264.7 macrophages. Results showed that ethanol extracts contained significant amount of polyphenols and chlorophylls, and possessed in vitro ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidative activities. The ethanol extracts did not show immunomodulatory activities in macrophages without lipopolysaccharide (LPS, 100 ng/ml) treatment, but significantly suppressed the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α, and such effects were associated with decreased expression of inducible NO synthase (iNOS) protein and cluster of differentiation 14 (CD14) mRNA, suggesting that NT2 has anti-inflammatory activities. On the other hand, the extracts increased matrix metalloproteinase (MMP)-9 activity, but showed no effects on Toll-like receptor (TLR) expression. In conclusion, ethanol extracts of NT2 possessed in vitro antioxidative activities and exhibited anti-inflammatory activities by decreasing NO, IL-6, and TNF-α production in association with decreased CD14 mRNA expression in LPS-induced RAW 264.7 macrophages, indicating its potential roles on health promotion and prentive effects against chronic diseases and such effects were and it’s in vitro antioxidant activities, suggesting its potential roles on health promotion.

Keywords: napiergrass, macrophage, lipopolysaccharide (LPS), anti-inflammation, antioxidation

Received July 01, 2020; Revised August 02, 2020; Accepted August 11, 2020

Citation: Yue-Hwa Chen, Yi-Han Wang, Yun-Ting Chen, Yuan-Yu Hou, Sassy Bhawamai, Yu-Wei Fang, Suppression of Inflammatory Mediators by Ethanol Extracts of Pennisetum purpureum S. (Napiergrass Taishigrass no. 2) in Activated RAW 264.7 Macrophages. Journal of Food and Nutrition Research, 8 (8). 392-398. January 2020. doi:10.12691/jfnr-8-8-2

1. Introduction

The immune system functions as one of the important lines of host defense against foreign pathogens in the body, and macrophages play significant roles in modulating immune functions. After being exposed to the bacterial endotoxin, lipopolysaccharide (LPS), for example, macrophages are activated to increase the production of various inflammatory mediators, including nitric oxide (NO), proinflammatory cytokines, such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α, as well as matrix metalloproteinases (MMPs). The membrane receptors, cluster of differentiation 14 (CD14) and Toll-like receptors (TLRs), and the transcription factor, nuclear factor (NF)-κB, were indicated to be involved in this activation process [1,2,3,4]. Under physiological conditions, these mediators are essential for antipathogenic processes and are well-regulated. However, dysregulated production of these mediators is linked to various pathological or oxidative conditions, such as a chronic inflammation status, autoimmune diseases, cardiovascular diseases, obesity, and cancers [5,6]. Therefore, compounds that possess antioxidative activities or suppress the overproduction of proinflammatory cytokines may play protective roles against inflammation and its associated diseases [7].

Napiergrass (Pennisetum purpureum S.) is widely used as livestock forage in Taiwan. Napiergrass Taishigrass no. 2 (NT2) is a new hybrid with shorter fibers and a better taste, so it is commonly used as one of the major ingredients in bioorganic energy soup. The bioorganic energy soup is a plant-based beverage, and is commonly consumed by healthy and illness pollutions, because it is thought to have health-promoting effects. Nowadays, scientific evidence regarding napiergrass has mainly focused on animal feeding [8,9] and renewable energy sources [10,11], but less information on its health-promoting effects was reported. Previously, the boiling-water extract of purple napiergrass was indicated to prevent in vitro low-density lipoprotein from oxidation, increase antioxidative enzyme activities in murine BNL hepatocytes, and decrease NO production in LPS-induced RAW 264.7 macrophages [12]. In addition, the boiling-water extract of NT2 also has in vitro antioxidative activities as determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the ferric-reducing antioxidant power (FRAP) [13]. Because only studies on boiling-water extract were reported, this study investigated the antioxidation and immunomodulation activities NT2, especially the ethanol extracts. Total polyphenol, coumaric acid, and chlorophylls contents, as well as in vitro antioxidant FRAP and DPPH of NT2 extracts were examined. The effects of ethanol extracts on immune-associated factors in both LPS-untreated and LPS-treated macrophages were also determined.

2. Materials and Methods

2.1. Chemicals and Biochemicals

RPMI 1640 medium, fetal bovine serum (FBS), and sodium bicarbonate were purchased from GIBCO BRL (Grand Island, NY, USA). Folin-Ciocalteau reagent, gallic acid, DPPH, LPS, absolute ethanol (≥ 99.8%), dimethyl sulfoxide (DMSO), sodium nitrite, and N-(1-naphthyl)ethylenediamine dihydrochloride were obtained from Sigma Chemical (St. Louis, MO, USA).

2.2. Preparation of NT2 Extracts

Freeze-dried NT2 powder purchased from Baoyuan Company (Taichung, Taiwan) was extracted with absolute ethanol or double-distilled water (ddH2O) (1:10 (w/v)) by a vertical rotating mixer for 24 h at room temperature. After centrifugation at 1600 xg, 4°C for 15 min, the supernatants were condensed with a rotary evaporator and adjusted to a final concentration of 1 or 2 g/ml with absolute ethanol or ddH2O, respectively. After being sterilized using 0.22-μm filters, the obtained ethanol and water extracts were used in the following experiments.

2.3. Determination of Total Phenols, Coumaric Acid, Chlorophylls, and in vitro Antioxidative Activities

Total polyphenol contents (TPCs) in NT2 extracts were determined according to methods described previously [14]. Briefly, the extracts were reacted with 50% Folin-Ciocalteau’s reagent for 10 min, followed by addition of 2% Na₂CO₃ for another 45 min, and the OD755 nm was determined. Gallic acid was served as a standard, and results are expressed as milligrams of gallic acid equivalents (GAE) per gram of dry NT2.

Being one of the most abundant polyphenols in NT2 [13], coumaric acid was determined by directly measuring the diluted extracts (1000 x dilution) at 305 nm due to its mesomeric effect of absorbing UV light at low concentrations (1~10 μM) [15], and purified coumaric acid (Sigma, St. Louis, MO, USA) served as a standard. Similar to coumaric acid, chlorophylls in the extracts were directly determined spectrophotometrically at wavelengths of 665 and 649 nm. Contents of chlorophylls a and b were calculated using the formulas 13.70 × A₆₆₅-5.76 × A₆₄₉ and -7.60 × A₆₆₅ + 25.8 × A₆₄₉, respectively [16].

To explore the antioxidative activities of the extracts, antioxidant power was measured by reacting it with a FRAP reagent followed by determining the absorbance at 593 nm using trolox as a standard [17], and results are expressed as trolox equivalents (TE) in mM/g dry weight. Free radical-scavenging activity was analyzed by the addition of a DPPH solution. After determining the absorbance at 515 nm, the scavenging ability was calculated according to the following formula [18]:

2.4. Cell Culture and Cell Proliferation

Murine RAW 264.7 macrophages were obtained from the Bioresource Collection and Research Center (BCRC #60001, Hsinchu, Taiwan). Cells were maintained in 10% FBS supplemented with RPMI 1640 medium at 37°C in a 5% CO₂ humidified atmosphere. After being seeded for 1 day, cells were changed to 1% FBS supplemented with RPMI 1640 medium and treated with various concentrations of ethanol extracts in the presence or absence of LPS (100 ng/ml) for 24 h. Absolute ethanol was added as a negative control, and ethanol added in the medium was 0.1%. Cells and media were then collected for analyses.

To evaluate the effects of the extracts on cell proliferation, cells (10⁴/well) were treated with NT2 extracts in the presence or absence of LPS for 24 h, and cell proliferation was determined using a CellTiter 96®Aqueous One Solution cell proliferation MTS assay kit (Promega, Madison, WI, USA).

2.5. Production of NO, Cytokines and MMPs

To understand the effects of NT2 on immune mediators, macrophages were treated with different concentrations of extracts in the presence or absence of LPS (100 ng/ml) for 24 h, and secretions of NO, IL-6, and TNF-α by macrophages were analyzed. The nitrite content was determined after adding the Griess reagent (1% sulfanilamide/0.1% naphthylethylene diaminedihydrochloride in 5% H₃PO₄) with absorbance at 590 nm [19]. IL-6 and TNF-α were detected by commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D System, Minneapolis, MN, USA) following the manufacturer’s instructions.

MMPs are a family of enzymes that are involved in extracellular matrix degradation, and were indicated to be associated with immune functions [20]. MMP activities were determined by a gelatin zymographic analysis. Briefly, culture medium (5 μl) was separated by 1 mg/ml gelatin containing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) [21]. Clear bands of MMP-2 (72 kDa) and MMP-9 (92 kDa) on the gel were observed after staining with 0.5% Coomassie blue R-250, and were quantified using the Image-Pro Plus software package (Media Cybernetics, Rockville, MD, USA).

2.6. Western Blot Analysis and Real-time Polymerase Chain Reaction (PCR)

To understand the involvement of iNOS protein in modulation of NO production, cell lysate containing 15 μg total cellular protein was separated by a 10% SDS-PAGE, and was transferred onto a polyvinylidene difluoride membrane. Expression of the iNOS protein was detected by adding anti-mouse iNOS (1:200) and anti-mouse β-actin (1:1000) antibodies followed by anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) and anti-mouse IgG-HRP (1:10000), respectively (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Images were observed after reacting with enhanced chemiluminescence (ECL) Western blotting detection reagent and exposure to chemiluminescent detection film. β-Actin served as an internal control.

To explore the effects of NT2 on mRNA expressions of immune-associated factors, total cellular RNA was extracted by the TRIzol® reagent (Ambion, Carlsbad, CA, USA), followed by reverse-transcription to complementary (c)DNA by reaction with oligo(dT)₁₂₋₁₈ and SuperScript® III reverse transcriptase (InvitrogenTM, Carlsbad, CA, USA). The obtained cDNA was used to conduct the PCR after adding various primers (Table 1) to an ABI 7300 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA).

Table 1. Primer sequences used in the RT-PCR

2.7. Statistical Analysis

All values are expressed as the mean ± standard deviation (SD), and the experiments were independently repeated at least 2 times. All statistical analyses were performed using SPSS vers. 19.0 (IBM, Armonk, NY, USA). A one-way analysis of variance (ANOVA) followed by post-hoc Tukey’s test was performed to compare differences between groups. A p value of <0.05 was considered significantly different.

3. Results

Total polyphenols and chlorophylls in both water and ethanol extracts were determined. Table 2 shows that higher levels of polyphenols, chlorophylls, and coumaric acid, one of the major phenolic compounds in NT2 [13], were detected in ethanol extracts. As expected, chlorophylls were not detected in water extracts due to its hydrophilicity. These results paralleled the antioxidant activities, in which higher FRAP and DPPH levels were observed in the ethanol extracts (Table 2). Therefore, the ethanol extracts were used in the cell culture experiments.

Results obtained from the MTS assay showed that the ethanol extracts of NT2, at concentrations ranging 0~2.5 mg/ml showed no effects on cell proliferation, regardless of the presence or absence of LPS. In the absence of LPS, cell proliferation even slightly increased (13% compared to the control group) at a concentration of 2.5 mg/ml (Figure 1).

Table 2. Polyphenol and chlorophyll contents, as well as in vitro antioxidant activities of napiergrass Taishigrass no. 2 (NT2) extracts

Figure 1. Effects of ethanol extracts of napiergrass Taishigrass no. 2 (NT2) on cell proliferation. RAW 264.7 macrophages were treated with various concentrations of extracts as indicated, in the absence or presence of LPS (100 ng/ml) for 24 h. Cell proliferation was measured by MTS assay. Values are means ± SDs (n = 3) from two independent experiments. ab, data that share no letters significantly differ by a one-way ANOVA (p < 0.05)

Figure 2. Effects of ethanol extracts of napiergrass Taishigrass no. 2 (NT2) on nitric oxide (NO) production (A) and inducible NO synthase (iNOS) protein expression (B) by RAW 264.7 macrophages. Cells were treated with different concentrations of extracts, in the presence or absence of lipopolysaccharide (LPS) (100 ng/ml), for 24 h. NO production was measured by the Griess reaction. iNOS protein expression was determined by Western blotting and the relative expressions were normalized to β-actin. Data are expressed as the mean ± SD from three independent experiments. * Significantly differs from the 0 (ethanol vehicle control) by Student’s t-test. ab, data that share no letters significantly differ by a one-way ANOVA (p < 0.05)

Figure 3. Effects of ethanol extracts of napiergrass Taishigrass no. 2 (NT2) on the production of interleukin (IL)-6 (A) and tumor necrosis factor (TNF)-α (B) by RAW 264.7 macrophages. Cells were treated with different concentrations of extracts, in the presence or absence of lipopolysaccharide (LPS) (100 ng/ml), for 24 h. Secretion of IL-6 and TNF-α into the cultured medium was analyzed by commercial assay kits according to the manufacturers’ instructions. Data are expressed as the mean ± SD from three independent experiments. * Significantly differs from the 0 (ethanol vehicle control) by Student’s t-test. abc, data that share no letters significantly differ by a one-way ANOVA (p < 0.05)

Figure 2 shows that the ethanol extracts did not affect NO production by macrophages, but concentration-dependently suppressed LPS-stimulated NO production by activated macrophages (Figure 2A). The decrease in NO production was mainly due to inhibition of iNOS expression, because stimulated expression of the iNOS protein was blocked by NT2 ethanol extracts (Figure 2B). Parallel to results obtained for NO, ethanol extracts did not affect the production of the proinflammatory cytokines, IL-6 or TNF-α, but significantly suppressed LPS-stimulated production of IL-6 and TNF-α in concentration-dependent manners (Figure 3). In addition to these immune mediators, the activity of MMP-9, but not MMP-2, was induced by LPS treatment, and was further enhanced by ethanol extracts, regardless of the presence of LPS (Figure 4). Being associated with LPS-stimulated inflammatory activities, the expressions of CD14 and TRL4 mRNAs were determined. As shown in Figure 5, NT2 ethanol extracts suppressed the mRNA expression of CD14, but showed no effects on TLR4 in LPS-activated macrophages.

Figure 4. Effects of ethanol extracts of napiergrass Taishigrass no. 2 (NT2) on matrix metalloproteinase (MMP) activities in RAW 264.7 macrophages. Cells were treated with different concentrations of extracts in the absence or presence of lipopolysaccharide (LPS) (100 ng/ml) for 24 h. The medium was collected to analyze MMP activities by gelatin zymography (A), and clear bands were quantified by Image-Pro Plus software (B, C). mRNA expression of MMP-9 was determined by a real-time PCR (D). Data are expressed as the mean ± SD from three independent experiments. a,b,#,*, data that share no symbols significantly differ within either LPS-untreated or LPS-treated groups by a one-way ANOVA (p < 0.05). MMP-2, p >0.05

Figure 5. Effects of ethanol extracts of napiergrass Taishigrass no. 2 (NT2) on mRNA expressions of cluster of differentiation 14 (CD14) (A) and Toll-like receptor 4 (TLR4) (B) by RAW 264.7 macrophages. Cells were treated with various concentrations of the extracts in the presence of lipopolysaccharide (LPS) (100 ng/ml) for 24 h. Total cellular RNA was isolated and analyzed for mRNA expression with a real-time PCR. The relative gene expression was obtained after normalization with GAPDH. Data are expressed as the mean ± SD (n = 3). ab, data that share no letters significantly differ by a one-way ANOVA (p < 0.05). TLR4, p > 0.05

4. Discussion

In the present study, we first demonstrated that the ethanol extracts of NT2 did not affect innate immunity, but possessed anti-inflammatory activities in stimulated macrophages. Large amounts of NO produced in response to the bacterial endotoxin, LPS, or cytokines play important roles in inflammation. Prolonged inflammation is a risk factor for various inflammatory diseases and pathological conditions, including rheumatoid arthritis, cardiovascular diseases, cancers, etc. [25,26]. In addition, high concentrations of NO may react with reactive oxygen species, including superoxide, produced by macrophages leading to the generation of peroxynitrite (ONOO¯), a powerful oxidant [27]. The induction of inflammatory responses by LPS is a receptor-mediated process [28]. After binding to complex membrane receptors CD14/TLR4, LPS initiates signaling transduction followed by activation of nuclear NF-κB and activator protein 1 (AP1) to increase the transcription of various immune mediators, such as iNOS, IL-6, and TNF-α [29]. Therefore, the inhibitory effects of NT2 ethanol extracts on LPS-stimulated NO, IL-6, and TNF-α production are, at least partially, attributed to the decrease in CD14 expression by macrophages, and may be mediated through modulating NF-κB's effects on AP-1 activities, although confirming this possibility requires further investigation. Although different studies [30,31] have shown that LPS induced TLR4 expression in RAW 264.7 macrophages, relatively low serum (1% FBS) and low LPS (100 ng/ml) concentrations used in the present study may explain the discrepancy. Alternatively, the possibility of direct interaction of the components in the extracts with LPS leading to its decreased bioactivity of LPS cannot be ruled out.

MMPs, on the other hand, are a group of proteases involved in extracellular matrix degradation, wound healing, tissue remodeling, and chemokine activation. Dysregulated MMP-9 has been linked to inflammatory responses, and its activity can be stimulated by LPS and proinflammatory cytokines [32]. However, pieces of evidence also indicate that MMP-9 presents anti-inflammatory effects by reducing inflammatory responses in skin tissues and by regulating cytokine production in immune cells [33]. Therefore, the NT2 ethanol extracts increased MMP-9 activity indicates the potential roles of NT2 on increasing our immune responses. However, NT2 further enhances the LPS-induced MMP-9 activity, similar to the results obtained from atorvastatin and simvastatin in RAW 264.7 macrophages. The clinical significance remains to be clarified.

Different active components, including chlorophylls and polyphenols, may contribute to the antioxidation and anti-inflammatory effects of NT2 ethanol extracts. Previously, Tsai et al. [13] indicated that polyphenols in boiling water extracts of NT2 play significant roles on its antioxidant activities. Among these, coumaric acid showed higher correlation to the antioxidant capacity than most other phenolic compounds in hot water extract, and its level is 10 X higher than the ethanol or cold water extracts detected in the present study. On the other hand, the chlorophylls were not determined in Tsai’s study due to its hydrophobic property. Therefore, chlorophylls are hypothesized as one of the potential bioactive components in ethanol extracts of NT2. Chlorophyll a was reported to suppress carrageenan-induced paw edema in mice and formalin-induced paw edema in rats [34], and inhibited LPS-stimulated NO production and thiobarbituric acid-reactive substances (TBARSs) in RAW 264.7 macrophages [35]. In addition, both chlorophylls a and b decreased TNF-α mRNA expression in LPS-induced HEK293 cells [34], and such an effect was associated with its in vitro antioxidant activity. On the other hand, coumaric acid, one of the major phenolic compounds in hot water extracts of NT2, decreased the TNF-α level in synovial tissue of rats with adjuvant-induced arthritis [36]. Rutin and quercetin, other than chlorophylls and coumaric acid, were also detected at relatively high levels in NT boiling water extract, and both compounds are able to inhibit TNF-α production and NO/iNOS protein expressions in activated RAW 264.7 macrophages [37,38]. These are all potential bioactive components in ethanol extracts of NT2 to suppress LPS-stimulated inflammatory mediators. In the present study, NT2 ethanol extracts added to culture medium contained 0.43~1.73 μg GAE/ml total polyphenols, 0.32~1.28 μg/ml coumaric acid, 0.66~2.63 μg/ml chlorophyll a, and 0.28~1.11 μg/ml chlorophyll b. Although concentrations of individual compounds in the culture medium were lower than those previously reported, these compounds may act additively or synergistically to inhibit inflammatory mediators in activated macrophages. In addition to NO and cytokines, different phytochemicals were shown to modulate MMP activities. Among these, lutein is one of the major carotenoids found in Triticum aestivum, a plant belongs to the same Poaceae family as NT [39], and it was indicated to increase MMP-9 activity and phagocytosis in RAW 264.7 macrophages [40]. As it can be extracted by ethanol, we propose that lutein may be one of the candidates which contribute to the induction of MMP-9 activity in NT ethanol extracts.

5. Conclusions

The present study suggests that ethanol extracts of NT2 possessed anti-inflammatory activities by decreasing the production of NO, IL-6, and TNF-α in LPS-activated RAW 264.7 macrophages, and such effects were associated with increased MMP-9 activity and decreased CD14 mRNA expression. The polyphenols and chlorophylls in the extracts may play roles as anti-inflammatory agents of NT2. Therefore, the consumption of NT2 mixed with nuts or oil, which is also usually added in the bioorganic energy soup, may have potential health-promoting effects. On the other hand, the ethanol extracts of NT2 showed no significant immunoregulatory effects on RAW 264.7 macrophages.

Acknowledgements

This research was funded by SHIN KONG WU HO-SU MEMORIAL HOSPITAL, TAIPEI, TAIWAN, grant number SKH-TMU-106-04.

Statement of Competing Interests

The authors have no competing interests.

List of Abbreviations

CD14 cluster of differentiation 14

DMSO dimethyl sulfoxide

DPPH 2,2-diphenyl-1-picrylhydrazyl

FBS fetal bovine serum

FRAP ferric-reducing antioxidant power

GAE gallic acid equivalents

HRP horseradish peroxidase

IL interleukin

iNOS inducible nitric oxide synthase

LPS lipopolysaccharide

MMP matrix metalloproteinase

NO nitric oxide

NT2 napiergrass Taishigrass no. 2

SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis

TE trolox equivalents

TLR Toll-like receptor

TNF tumor necrosis factor

TPCs total polyphenol contents

References

[1]   | Baldwin, A.S., Jr. “The NF-kappa B and I kappa B proteins: new discoveries and insights.” Annual Review of Immunology, 14 (1). 649-681, Apr. 1996.
[2]   | Rhee, J.W., Lee, K.W., Kim, D., Lee, Y., Jeon, O.H., Kwon, H.J. and Kim, D.S. “NF-kappaB-dependent regulation of matrix metalloproteinase-9 gene expression by lipopolysaccharide in a macrophage cell line RAW 264.7.” Journal of Biochemisty and Molecular Biology, 40 (1). 88-94. Jan. 2007.
[3]   | Guo, J. and Friedman, S.L. “Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis.” Fibrogenesis Tissue Repair, 3 (1). 21. Oct. 2010.
[4]   | Murray, P.J. and Wynn, T.A. “Protective and pathogenic functions of macrophage subsets.” Nature Reviews Immunology, 11 (11). 723-737. Oct. 2011.
[5]   | Linker-Israeli, M., Deans, R.J., Wallace, D.J., Prehn, J., Ozeri-Chen, T. and Klinenberg, J.R. “Elevated levels of endogenous IL-6 in systemic lupus erythematosus. A putative role in pathogenesis.” The Journal of Immunology, 147 (1). 117-123. July 1991.
[6]   | Shrivastava, A.K., Singh, H.V., Raizada, A., Singh, S.K., Pandey, A., Singh, N., Yadav, D.S. and Sharma, H. “Inflammatory markers in patients with rheumatoid arthritis.” Allergologiaet Immunopathologia, 43 (1). 81-87. Jan-Feb. 2015.
[7]   | Mueller, M., Hobiger, S. and Jungbauer, A. “Anti-inflammatory activity of extracts from fruits, herbs and spices.” Food Chemistry, 122(4), 987-996. Oct. 2010.
[8]   | Rahman, M.M.; Abdullah, R.B.; Wan Embong, W.K.; Nakagawa, T.; Akashi, R. “Effect of palm kernel cake as protein source in a concentrate diet on intake, digestibility and live weight gain of goats fed Napier grass.” Tropical Animal Health Production, 45 (3). 873-878. Mar. 2013.
[9]   | Wanjala, B.W., Obonyo, M., Wachira, F.N., Muchugi, A., Mulaa, M., Harvey, J., Skilton, R.A., Proud, J. and Hanson, J. “Genetic diversity in Napier grass (Pennisetum purpureum) cultivars: implications for breeding and conservation.” AoB Plants, 5. plt022. May 2013

[10]  
Scholl, A.L., Menegol, D., Pitarelo, A.P., Fontana, R.C., Filho, A.Z., Ramos, L.P., Dillon, A.J. and Camassola, M. “Ethanol production from sugars obtained during enzymatic hydrolysis of elephant grass (Pennisetum purpureum, Schum.) pretreated by steam explosion.” Bioresource Technology, 192, 228-237. Sep. 2015.

[11]   | Takara, D. and Khanal, S.K. “Characterizing compositional changes of Napier grass at different stages of growth for biofuel and biobased products potential.” Bioresource Technology, 188, 103-108. Jul. 2015.
[12]   | Yu, H.M., Wang, B.-S., Chu, H.L., Chang, L.-W., Yen, W.-J., Lin, C.-J. and Duh, P.-D. “Napiergrass (Pennisetum purpureum S.) protects oxidative damage of biomolecules and modulates antioxidant enzyme activity.” Food Chemistry, 105 (4). 1364-1374. 2007.
[13]   | Tsai, P.-J., Wu, S.-C. and Cheng, Y.-K. “Role of polyphenols in antioxidant capacity of napiergrass from different growing seasons.” Food Chemistry, 106 (1). 27-32. Jan. 2008.
[14]   | Cicco, N., Lanorte, M.T., Paraggio, M., Viggiano, M. and Lattanzio, V. “A reproducible, rapid and inexpensive Folin-Ciocalteu micro-method in determining phenolics of plant methanol extracts.” Microchemical Journal, 91 (1). 107-110. Jan. 2009.
[15]   | Barthelmebs, L., Divies, C. and Cavin, J.F. “Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism.” Applied and Environmental Microbiology, 66 (8). 3368-3375. Aug. 2000.
[16]   | Ritchie, R.J. “Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents.” Photosynthesis Research, 89, 27-41. Jun. 2006.
[17]   | Benzie, I.F.F. and Strain, J.J. “The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay”. Analytical Biochemistry, 239 (1). 70-76. Jul.1996.
[18]   | Sharma, O.P. and Bhat, T.K. “DPPH antioxidant assay revisited.” Food Chemistry, 113(4). 1202-1205. Apr. 2009.
[19]   | Privat, C., Lantoine, F., Bedioui, F., Millanvoye van Brussel, E., Devynck, J. and Devynck, M.A. “Nitric oxide production by endothelial cells: comparison of three methods of quantification.” Life Sciences, 61 (12). 1193-1202. Aug. 1997.
[20]   | Parks, W.C., Wilson, C.L. and Lopez-Boado, Y.S. “Matrix metalloproteinases as modulators of inflammation and innate immunity.” Nature Reviews Immunology, 4 (8), 617-629. Aug. 2004.
[21]   | Birkedal-Hansen, H. and Taylor, R.E. “Detergent-activation of latent collagenase and resolution of its component molecules.” Biochemical and Biophysical Research Communications, 107 (4), 1173-1178. Aug. 1982.

[22]  
Choi, H.J., Chung, T.W., Kim, J.E., Jeong, H.S., Joo, M., Cha, J., Kim, C.H. and Ha, K.T. “Aesculin inhibits matrix metalloproteinase-9 expression via p38 mitogen activated protein kinase and activator protein 1 in lipopolysaccharide-induced RAW264.7 cells.” International Immunopharmacology, 14 (3). 267-274. Nov. 2012.

[23]   | Wang, J.X., Hou, L.F., Yang, Y., Tang, W., Li, Y. and Zuo, J.P. “SM905, an artemisinin derivative, inhibited NO and pro-inflammatory cytokine production by suppressing MAPK and NF-kappaB pathways in RAW 264.7 macrophages.” Acta Pharmacologica Sinica, 30 (10), 1428-1435. Oct. 2009.
[24]   | Yeo, S.J., Yoon, J.G., Hong, S.C. and Yi, A.K. ”CpG DNA induces self and cross-hyporesponsiveness of RAW264.7 cells in response to CpG DNA and lipopolysaccharide: alterations in IL-1 receptor-associated kinase expression.” Journal of Immunology, 170 (2), 1052-1061. Jan. 2003.
[25]   | Whicher, J. T. and Evans, S. W. “Cytokines in disease.” Clinical Chemistry, 36 (7), 1269-1281. Jul. 1990.
[26]   | Ohshima, H. and Bartsch, H. “Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis.” Mutation Research, 305 (2), 253-264. Mar.1994.
[27]   | Tamir, S.; Tannenbaum, S. R. “The role of nitric oxide (NO) in the carcinogenic process.” Biochimica et Biophysica Acta, 1288(2), F31-F36. Oct. 1996.
[28]   | Fujihara, M., Muroi, M., Tanamoto, K., Suzuki, T., Azuma, H. and Ikeda, H. “Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex.” Pharmacology & Therapeutics, 100 (2), 171-194. Nov. 2003.
[29]   | Aderem, A. and Ulevitch, R.J. “Toll-like receptors in the induction of the innate immune response.” Nature, 406 (6797), 782-787. Aug. 2000.
[30]   | Premkumar, V., Dey, M., Dorn, R. and Raskin, I. “MyD88-dependent and independent pathways of Toll-Like Receptors are engaged in biological activity of Triptolide in ligand-stimulated macrophages.” BMC Chemical Biology, 10. 3. Apr. 2010.

[31]  
Yang, Y., Li, S., Yang, Q., Shi, Y., Zheng, M., Liu, Y., Chen, F., Song, G., Xu, H., Wan, T., He, J. and Chen, Z. “Resveratrol reduces the proinflammatory effects and lipopolysaccharide-induced expression of HMGB1 and TLR4 in RAW264.7 cells.” Cellular Physiology and Biochemistry, 33 (5). 1283-1292. Apr. 2014.

[32]   | Nissinen, L. and Kähäri, V.-M. “Matrix metalloproteinases in inflammation.” Biochimica et. Biophysica Acta 1840.(8). 2571-2580. Aug. 2014.

[33]  
Lee, D.K., Park, E.J., Kim, E.K., Jin, J.Y., Kim, J.S., Shin, I.J., Kim, B.Y., Lee, H. and Kim, D.-E. “Atorvastatin and simvastatin, but not pravastatin, up-regulate LPS-induced MMP-9 expression in macrophages by regulating phosphorylation of ERK and CREB.” Cellular Physiolology and Biochemistry, 30 (3). 499-511. July 2012.

[34]  
Subramoniam, A., Asha, V.V., Nair, S.A., Sasidharan, S.P., Sureshkumar, P.K., Rajendran, K.N., Karunagaran, D. and Ramalingam, K. “Chlorophyll revisited: anti-inflammatory activities of chlorophyll a and inhibition of expression of TNF-alpha gene by the same.” Inflammation, 35 (3), 959-966. Jun. 2012.

[35]   | Park, J.-Y., Park, C.-M., Kim, J.-J., Noh, K.-H., Cho, C.-W. and Song, Y.-S. “The protective effect of chlorophyll a against oxidative stress and inflammatory processes in LPS-stimulated macrophages.” Food Science and Biotechnology, 16 (2), 205-211. Apr. 2007.
[36]   | Pragasam, S.J., Venkatesan, V. and Rasool, M. “Immunomodulatory and anti-inflammatory effect of p-coumaric acid, a common dietary polyphenol on experimental inflammation in rats.” Inflammation, 36 (1). 169-176. Feb. 2013.
[37]   | De Stefano, D., Maiuri, M.C., Simeon, V., Grassia, G., Soscia, A., Cinelli, M.P. and Carnuccio, R. “Lycopene, quercetin and tyrosol prevent macrophage activation induced by gliadin and IFN-gamma.” European Journal of Pharmacology, 566 (1-3), 192-199 Jul. 2007.
[38]   | Gao, M., Ma, Y. and Liu, D. “Rutin suppresses palmitic acids-triggered inflammation in macrophages and blocks high fat diet-induced obesity and fatty liver in mice.” Pharmaceutical Research, 30 (11), 2940-2950. Nov. 2013.
[39]   | O’Sullivan, A.M., O’Callaghan, Y.C., O’Connor, T.P. and O’Brien, N.M. “The content and bioaccessibility of carotenoids from selected commercially available health supplements.” Proceedings of the Nutrition Society, 70 (OCE3), E62. Aug. 2011.

[40]  
Lo, H.M., Chen, C.L. Yang, C.M., Wu, P.H., Tsou, C.J., Chiang, K.W. and Wu, W.B. “The carotenoid lutein enhances matrix metalloproteinase-9 production and phagocytosis through intracellular ROS generation and ERK1/2, p38 MAPK, and RARbeta activation in murine macrophages.” Journal of Leukocyte Biology, 93 (5), 723-735. May 2013.