Immunostimulatory Activity Investigation of Aqueous and Hydroethanolic Extract of Wheatgrass Using THP1 Cells

MOJ Immunology

Research Article Abstract Volume 5 Issue 1 - 2017 Wheatgrass, Triticum aestivum is well known for its high nutritional values. However the immunomodulatory activity of the same is unexplored. Therefore, the present study was planned to elucidate the immunomodulatory activity of aqueous and hydro-ethanolic extract of wheatgrass. For immunomodulatory activity analysis, THP1 cells were treated with and without lipopolysaccharide Defence Institute of Physiology & Allied Sciences (DIPAS), India (LPS) and aqueous and hydroethanolic wheatgrass extract (50µg/mL and 100µg/ mL, respectively) for 24 h. After 24 h, supernatants were harvested and cytokine *Corresponding author: Richa Rathor, Defence Institute assays were performed. NO production in terms of amount of nitrite content of Physiology & Allied Sciences (DIPAS), Lucknow Road, was also estimated in treated and untreated murine peritoneal macrophages Timarpur, Delhi-110054, India, Tel: 91-11-23946257; Fax: using Griess reagent. Further, a dose dependent in-vitro comparative study 91-11-23914790; Email: concluded that both the extracts possess immunostimulatory activity. However comparison between both established that aqueous extract of wheatgrass Received: December 06, 2016 | Published: January 24, 2017

cells.having The more extract immunostimulatory also significantly activity induced at the nitrite dose release of 100μg/ml in macrophage as extract cellssignificantly without upregulated causing any LPS toxic induced effects. release The findings of TNF-α, suggest IL-1β andthe IL-6non-specific in THP1 immunostimulatory activity of wheatgrass; hence it could be used in enhancing immune responses and disease resistance.

Keywords: Immunomodulation; Wheatgrass; Triticum aestivum; Cytokines; THP1 cells; Lipopolysaccharide; Immune responses; Disease resistance; THP1 cells

Abbreviations: WG1: Aqueous Extract of Wheatgrass; observed towards the use of plant/herbal products as an effective WG2: Hydro-Ethanolic Extract of Wheatgrass; C: Control; LPS: alternative therapy for the prevention and treatment of numerous Lipopolysaccharide; FBS: Fetal Bovine Serum; PBS: Phosphate medical problems as plant products have advantages over the conventionally used drugs, which are not only expensive but also

NationalBuffered CentreSaline; forNO: Cell Nitric Sciences Oxide; TNF-α: Tumor Necrosis Factor- α; DMSO: Dimethyl Sulfoxide; ANOVA: Analysis of Variance; NCCS: haveWheatgrass harmful side basically effects [5,6].is young grass of the common wheat Introduction plant that belongs to Poaceae family. Commonly, it is edible in India, prepared from cotyledons of the common wheat plant, Immunomodulation means the ability to alter immune Triticum aestivum. Triticum aestivum may be consumed as fresh system of an organism via interfering its functions. Enhancement of immune system is known as immunostimulation which wheatgrass is having full nutritional value which is cause of having antioxidant,juice or dried hypocholestrolemic,powder by animal and anti-carcinogenic, human. It is believed laxative, that the other hand, immunosuppressant implies reduction of resistanceprimarily impliesagainst infections stimulation and of stress. non-specific Immunostimulation system; on Beside these properties, wheatgrass is also used for number of and immunosuppression both activities require for regulating conditionsastringent, diuretic,like common antibacterial cold, cough, and anti-agingbronchitis, properties fever, infections, [7-10]. the normal immunological functioning. Therefore both immunostimulating and immunosuppressing have their own importance and search for better agents exerting these activities inflamed mouth and throat, and skin disorders like hemorrhoids, haspsoriasis, commenced ivy, eczema, to provide burns its andimmunomodulatory thalassemia [11]. activity. In spite To keepingof having these numerous facts in beneficialour mind, properties,the present acomparative limited research study From the ancient time, plant products were used as is becoming the field of major interest all over the world [1-2]. was planned to investigate the immunomodulatory activity of immunostimulant or immunosuppressant for cure and remedy of aqueous and hydro-ethanolic extract of wheatgrass using humans. Beside this, these plant based product also used as the in-vitro model, LPS-stimulated human monocytic THP1 cells. principal content drugs [3-4]. Recently, a growing attraction was

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Materials and Methods Chemicals was collected and stored at -80°C until measurements of TNF-α, IL-6 and IL-1β was performed. RPMI and fetal bovine serum (FBS) were purchased from using commercial enzyme-linked immunosorbent assay (ELISA) kitsTNF-α, (Cayman, IL-6 USA) and IL-1β following levels manufacturer’s in supernatant instructions.were determined by purchased from Cayman Chemicals, New Orleans, Louisiana, USA. LipopolysaccraideHiMedia, India. TNF-α, (LPS) IL-1β,was purchased IL-6 and from NF-κB Sigma, ELISA USA. kits were Mouse peritoneal macrophages isolation and culture for measurement of nitrite concentration Plant material To study the effects of wheatgrass extract on NO production, Wheatgrass was procured from Grime’s WG, Sholapur, a Maharashtra, India. The characterized commercially available measured as nitrite released from mouse macrophage cells. wheatgrass was used in the present study. Leaves of wheatgrass For griess isolation reagent of macrophages based method cells, was used2% starch [13]. Nitricsuspension oxide was was clean twice with distilled water followed by it was dried in prepared in phosphate buffered saline (PBS) and 1.5 ml of the clean, shaded and dust free area. Dry leaves were crushed by laboratory blander and further used for extraction process. above solution was injected into the peritoneal cavity of mice. Preparation of aqueous and hydro-ethanolic extract of 400gAfter 3for days, 10 min; peritoneal the resulting fluid was pellet collected was suspended in the cold in phosphate complete wheatgrass RPMI1640buffer saline medium (PBS). Collectedat a concentration peritoneal of fluid 5×10 was5 cells centrifuged per ml. The at Accelerated solvent Extraction system ASE 350 equipped with cells are allowed to adhere to the substrate by culturing them 2 hr a solvent controller unit from Dionex Corporation (Sunnyvale, CA, at 37°C. Nonadherent cells are removed by gently washing three USA) was used for the extraction. Extraction was carried in 33 times with warm PBS. At this time, total cell population was 92%. ml extraction cells, containing 2gm of sample in triplicate using Mouse macrophages were cultured in RPMI1640 media with millipore water and 70% ethanol as a solvent (for aqueous and 25mM HEPES, 2mM glutamine, 10% fetal bovine serum, penicillin hydro-ethanolic extract respectively) at 25°C±2°C for 15 min. The (100 IU/ml), and streptomycin (100 IU/ml). Mouse peritoneal heat-up time changed according to extraction temperature and is macrophages were cultured with different concentrations of aqueous and hydro-ethanolic extract of wheatgrass with or for automaticallyIn vitro Immunomodulatory fixed by the equipment. activity 2 48hrs. NO production in supernatant was measured in terms Cell Line and Tissue Culture Media: THP1 cell line was obtained ofwithout amount LPS of innitrite, a humidified its stable incubator product atin 37°Ca microplate in 5% CO assay. from National Centre for Cell Sciences (NCCS), Pune, India. Cells were grown in RPMI 1640 media containing 25mM HEPES, 2mM was collected, mixed with an equal volume of Griess reagent (A l-glutamine (Sigma, St. Louis, MO), 10% heat inactivated fetal solutionTo measure of sulfanilamide nitrite, 100 μL and of macrophageN-(1-naphthyl)-ethylenediamine culture supernatant bovine serum, penicillin (100 U/ml), and streptomycin (100ug/ in 2M hydrochloric acid) and incubated for 10 min at room at 37°C. 2 temperature. Nitrite concentration was determined by measuring the absorbance at 550 nm with a microplate reader. NaNO was ml)MTT in Assaya humidified for cell atmosphere viability of 5% CO used for external calibration. The cell viability was tested using yellow tetrazolium salt, Nuclear transcription factor – kB expression (NF-kB) 5 plates. In brief, THP-1 cells (2X10 cells/ml) were incubated 1.0X107cells/ml was suspended in RPMI 1640, centrifuged at MTT [12]. The assay was performed in 96-well tissueat 37°C culture with 2 300Xg for 5 min at 4°C and the supernatant was discarded. Then, varying concentration of aqueous and hydro-ethanolic extract nuclear extract was prepared as per instructions given in Cayman’s for 24 h in a humidified atmosphere of 5% CO of wheatgrass in the presence and absence of LPS (0.2 µg/ml). Nuclear Extraction Kit, U.S.A. Then NF-kB was estimated by ELISA After incubation, 20 µl of 5mg/ml MTT solution was added kit (Cayman, USA). All tests were performed in triplicates. and incubated for additional 4hr under the same conditions. Followed by, the supernatant were aspirated to an eppendorf Statistical analysis tube from culture plate and centrifuged at 3200rpm at 4°C for 15 Data are presented as Mean ± SEM. One way Analysis of min. Supernatant was removed and the blue formazon crystals Variance (ANOVA) followed by Berferroni’s multiple comparisons were solubilized in 200 µl of dimethyl sulfoxide (DMSO) under test using Graph Pad Instat7 (Graph Pad Software Inc, La Jolla, agitation. After dissolving the crystals, optical density were in a microtiter plate reader (BioTek, Powerwave XS2, USA) at 570 nm. Determination of TNF-α, IL-6 and IL-1β ResultsCA). A p-value <0.05 was considered statistically significant. THP-1 cells (2X105cells/ml) were incubated with different In this study, human monocyte cell line was used to identify concentrations of aqueous and hydro-ethanolic extract of the immunomodulatory activity of wheatgrass (different extract wheatgrass in the absence and presence of LPS (0.2µg/ml) for 24 and different concentration) using various immunomodulatory

2 at 37°C. Supernatant hrs in a humidified atmosphere of 5% CO markers (proinflammatory cytokines, TNF-α, IL-1β and IL-6) in unstimulated and LPS-stimulated THP-1 cells [14].

Citation: Rathor R, Meena DK, Shyam R, Misra K (2017) Immunostimulatory Activity Investigation of Aqueous and Hydroethanolic Extract of Wheatgrass Using THP1 Cells. MOJ Immunol 5(1): 00146. DOI:

Effect of aqueous and hydro-ethanolic extract of wheatgrass on cell viability (59.07±3.868pg/ml, p<0.05) (Figure 3). this, WG1 alone at the dose of 100µg/ml also increased IL-1β In the same experiment, we also evaluated if the stimulatory effect on cytokine release due to direct upregulated effect of THP1 cells by aqueous and hydro-ethanolic extract of wheatgrass. The viability of THP1 cells were measured by MTT assay. stimulated and wheat grass treated cells which demonstrated that viability No was significant unaffected change by using was aqueous observed and in hydro-ethanolic unstimulated, extract of wheatgrass (Figure 1).

Figure 2: Effect of aqueous and hydro-ethanolic extract of wheatgrass

onWG1: the productionAqueous ofextract TNF-α ofin THP1wheatgrass; cells. WG2: Hydro-ethanolic extract of wheatgrass; C + LPS: Lipopolysaccharide treated; L+WG1: Lipopolysaccharide+Aqueous extract of wheatgrass treated; L+WG2: Lipopolysaccharide+Hydro-ethanolic extract of wheatgrass treated. *,†,‡,§Differences between values with matching symbol notations Figure 1: Effect of aqueous and hydro-ethanolic extract of wheatgrass cell viability in THP1 cells. probability. Data represents Means±SEM of three independent experimentswithin each carried column out are in not triplicates. statistically significant at 5% level of WG1: Aqueous extract of wheatgrass; WG2: Hydro-ethanolic extract of wheatgrass; C+LPS: Lipopolysaccharide treated; L+WG1: Lipopolysaccharide +Aqueous extract of wheatgrass treated; L+WG2: Lipopolysaccharide+Hydro-ethanolic extract of wheatgrass treated. Data represents Means±SEM of three independent experiments carried out in triplicates.

Effect of aqueous and hydro-ethanolic extract of wheatgrass on TNF-α release plays roles in antitumor activity, immune modulation, Tumor necrosis factor-α (TNF-α), also known as cachectin, inflammation, anorexia, cachexia, septic shock, viral replication, and hematopoiesis [15,16]. The results depicted TNF- α onceproduction cells were increased treated with significantly LPS and WG1 in LPSat the treated dose of cells100 µg/ in mlcomparison (2756±48.87pg/ml, to control cells.p<0.05 TNF-). Beside α secretion this, WG1 was alone also increased (100 µg/ Figure 3: Effect of aqueous and hydro-ethanolic extract of wheatgrass

(Figure 2). onWG1: the productionAqueous ofextract IL-1β inof THP1 wheatgrass; cells. WG2: Hydro-ethanolic ml) also significantly increased TNF-α level (358±10.42pg/ml) extract of wheatgrass; C + LPS: Lipopolysaccharide treated; L+WG1: Effect of aqueous and hydro-ethanolic extract of Lipopolysaccharide +Aqueous extract of wheatgrass treated; L+WG2: wheatgrass on IL-1β release Lipopolysaccharide+Hydro-ethanolic extract of wheatgrass treated. *,†,‡,§Differences between values with matching symbol notations comparison to control (36.94±0.61 pg ml and 288.6±8.91pg/ml respectively).IL-1β significantly WG1 extract increased at the dose in LPS of 50 treated and 100 THP1 µg/ml cells was in probability. Data represents Means±SEM of three independent experimentswithin each carried column out are in not triplicates. statistically significant at 5% level of able to increase more IL-1β secretion in LPS treated cells. Beside

Citation: Rathor R, Meena DK, Shyam R, Misra K (2017) Immunostimulatory Activity Investigation of Aqueous and Hydroethanolic Extract of Wheatgrass Using THP1 Cells. MOJ Immunol 5(1): 00146. DOI:

Effect of aqueous and hydro-ethanolic extract of Effect of aqueous and hydro-ethanolic extract of wheatgrass on IL-6 release wheatgrass on NF-κB expression

THP1 cells compared with control cells (5.23±0.472pg/ml and With the support of other parameters, the expression of the same 212.3±6.339pg/mlA significant increment respectively). of IL-6 The was increase observed of inIL-6 LPS was treated also NF-κB expression increased significantly in LPS exposed cells.

increase significantly in cells treated with higher dose of WG1 increased significantly once cells were only treated with WG1 (100μg/ml) with or without LPS (Figure 5). (100 μg/ml) (Figure 4).

Figure 4: Effect of aqueous and hydro-ethanolic extract of wheatgrass Figure 5: Effect of aqueous and hydro-ethanolic extract of wheatgrass on the production of IL-6 in THP1 cells.

WG1: Aqueous extract of wheatgrass; WG2: Hydro-ethanolic onWG1: the productionAqueous ofextract NF-κB ofin THP1wheatgrass; cells. WG2: Hydro-ethanolic extract of wheatgrass; C + LPS: Lipopolysaccharide treated; L+WG1: extract of wheatgrass; C + LPS: Lipopolysaccharide treated; L+WG1: Lipopolysaccharide +Aqueous extract of wheatgrass treated; L+WG2: Lipopolysaccharide +Aqueous extract of wheatgrass treated; L+WG2: Lipopolysaccharide+Hydro-ethanolic extract of wheatgrass treated. Lipopolysaccharide+Hydro-ethanolic extract of wheatgrass treated. *,†,‡,§Differences between values with matching symbol notations *,†,‡Differences between values with matching symbol notations

probability. Data represents Means±SEM of three independent probability. Data represents Means±SEM of three independent experimentswithin each carried column out are in not triplicates. statistically significant at 5% level of experimentswithin each carried column out are in nottriplicates. statistically significant at 5%level of

Effect of aqueous and hydro-ethanolic extract of Discussion wheatgrass on Nitrite production The present study gives an overview of immunomodulatory activity of wheatgrass, a popular herb of India with special THP1 cells compared with control cells. The increase in NO was Nitrite production was increased significantly in LPS treated reference to pro-inflammatory cytokines (viz TNF-α, IL-1β and increased more after treatment with 100μg/ml of WG1 (Figure 6). wheatgrassIL-6 expression) and described and NF-κB the activitypresence using of gallic monocytic acid and cellrutin line as nutritionalTHP1. Khan supplement et al. [17] conductedin the same a extract. chromatographic analysis of The present study made a comparison between aqueous and hydro-ethanolic extract of wheat grass in view of immunomodulatory activity. Interestingly, we found that both the extract possessed immunostimulatory activity but aqueous extract of wheatgrass was having more immunostimutory activity as compared with hydro-ethanolic extract of wheatgrass.

Figure 6: Effect of aqueous and hydro-ethanolic extract of wheatgrass The first response towards infection is inflammation which on nitrite production in THP1 cells. theseused tocytokines start with could the production be considered of pro-inflammatory as immune-suppressive cytokines WG1: Aqueous extract of wheatgrass; WG2: Hydro-ethanolic druglike TNF-α,while enhancers IL-1β and of nitric these oxide cytokines (NO). could Thus be inhibitors known as of extract of wheatgrass; C + LPS: Lipopolysaccharide treated; L+WG1: immune-stimulators. The widely distributed key mediators of Lipopolysaccharide +Aqueous extract of wheatgrass treated; L+WG2: Lipopolysaccharide+Hydro-ethanolic extract of wheatgrass treated. this, endotoxin or other external stimuli activates monocytes/ *,†,‡,§Differences between values with matching symbol notations inflammation are monocytes/macrophages [18]. Along with

probability. Data represents Means±SEM of three independent macrophages which also engage to produce pro-inflammatory experimentswithin each carried column out are in not triplicates. statistically significant at 5% level of cytokines like TNF-α, IL-1β and IL-6 [19]. Beside the cytokines cascade, other inflammatory mediators such nitric oxide (NO) and NF-kB also play an important role during inflammation

Citation: Rathor R, Meena DK, Shyam R, Misra K (2017) Immunostimulatory Activity Investigation of Aqueous and Hydroethanolic Extract of Wheatgrass Using THP1 Cells. MOJ Immunol 5(1): 00146. DOI:

wheatgrass. Beside this immune-stimulant role of wheatgrass

[20,21]. Nitric oxide (NO) is a signaling molecule which used to was observed in MTT assay at any dose of wheatgrass extract play a prime role in pathogenesis of inflammation. Under normal onwas LPS not stimulated associated andwith unstimulated any cell death THP1 as no cells. significant In other change word, condition, it gives anti-inflammatory effect while during abnormal it could also be state that cell viability was not affected due to situation, it used to consider as pro-inflammatory mediator that wheatgrass treatment (Figure 7). Conclusively, the present study ininduces regulating inflammation, various genes vasoconstriction for cytokines, and chemokines, tissue damage adhesion [22]. Nuclear factor kappa B (NF-κB), a transcription factor involved responsible for growth, development, apoptosis and immune/ depicts non-specific immunostimulatory activity of wheatgrass. targets and acute phase proteins. Beside this, NF-κB is also Non-specific immunostimulant used to enhance innate or non-specific immune response via interacting and activating immunomodulatory responses [21]. The present results showed immune cells. Another well known fact regarding non-specific oxideupregulation and NF-kB of pro-inflammatory was also found increased cytokine during levels suchaqueous TNF-α, wheat IL- autoimmunityimmunostimulants and thatneoplastic it do not diseases. contain Immunostimulantsantigen specific activity can grass1β and extract IL-6. Along treatment. with this, nitrite production, indicator of nitric beand grouped are widely under used different in chronic agents infections, based on the immunodeficiency, source, such as bacterial preparations, polysaccharides, animal or plant extracts,

wheatgrass could also be used in enhancing immune responses LPS-stimulatedThe finding of THP1 the present cells. Moreover, study demonstrated this induction that wheatgrassof immune andnutritional disease factorsresistance. and cytokines [23,24]. Based on these facts is a potent activator of TNF-α, IL-1β, IL-6 and nitrite production in response was also associated with NF-κB activation due to

Figure 7: Graphical Representation of experimental procedure for analysis of immunostimulatory activity of wheat grass.

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Citation: Rathor R, Meena DK, Shyam R, Misra K (2017) Immunostimulatory Activity Investigation of Aqueous and Hydroethanolic Extract of Wheatgrass Using THP1 Cells. MOJ Immunol 5(1): 00146. DOI:

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Citation: Rathor R, Meena DK, Shyam R, Misra K (2017) Immunostimulatory Activity Investigation of Aqueous and Hydroethanolic Extract of Wheatgrass Using THP1 Cells. MOJ Immunol 5(1): 00146. DOI:

10.15406/moji.2017.05.00146