TJPS Vol.41 (Supplement Issue) 2017

TJPS 2017, 41 (Supplement Issue): 217

Anti-inflammatory activities of Thai rice bran extracts in

fibroblast and macrophage cells

Warintra Sawangsri1, Primchanien Moongkarndi2, Malyn Ungsurungsie3, Boonchoo Sritularak4

Supachoke Mangmool1,*

1Department of Pharmacology; 2Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand 3Research and development division, S&J International Enterprises Public Company Limited, Bangkok 10120, Thailand 4Department of Pharmacognosy and Pharmaceutical Botany; Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand

* Corresponding author: Tel. +66(0)840017654; Fax. +66(0)26448700; E-mail address: supachoke.man@mahidol.ac.th

Keywords: Anti-inflammatory activity / Inflammatory mediator / L929 / Raw 264.7 / Rice bran

Introduction The inflammation is the mechanism that involves with immune systems by release and synthesis of

several inflammatory mediators1. Cells in skin such as keratinocytes, fibroblasts, and macrophages produce

inflammatory mediators after stimulated that cause chronic inflammatory skin diseases and aging2. Rice bran

contains several compounds that have health-supportive properties including anti-inflammation3. However, the

anti-inflammatory activities of Thai pigmented rice bran extracts in the cells have been still unclear. In this

study, we investigated the effects of Thai pigmented rice bran extracts including Red Hawm, Red Rose, and

Black Sukhothai II on LPS-induced the synthesis and secretion of inflammatory mediators in L929 murine

fibroblast cells and RAW 264.7 murine macrophage cells.

Methods Chemicals and reagents: Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin 0.25% EDTA, fetal bovine

serum (FBS), penicillin-streptomycin solution, MTT solution, DMSO were purchased from Biowest Co. (NY,

MO, USA), Jena Bioscience Co. (Germany), and Roche (Germany). Lipopolysaccharide (LPS) from

Salmonella enterica serotype typhimurium and indomethacin were purchased from Sigma-Aldrich (St, Louise,

MO, USA). Three Thai rice bran extracts, Red Hawm, Red Rose, and Black Sukhothai II, were generous gifts

from Prof. Malyn Ungsurungsie (Research and development division, S&J International Enterprises Public

Company, Thailand).

Cell culture: L929 and RAW 264.7 cells were cultured in DMEM with 10% FBS and penicillin-streptomycin

solution diluted 1:100 and incubated in a humidified atmosphere of 5% CO2 incubator under temperature

37°C. Cell were grown in culture dishes and passaged by trypsinization every 3-5 days.

Cell toxicity testing by using MTT assay: L929 cells and RAW 264.7 cells were seeded in 96-well culture

plates at a 1.0 x 104 cells/well density in the 200 µl of DMEM supplemented with 1% FBS and 1% P/S and

incubated for 24 hours. After incubation, the cultured cells were treated with and without the varied

concentrations of Thai rice bran extracts (0.01-5 mg/ml, diluted with DMEM) and then incubated for 36 hours.

The culture medium was replaced with 200 µl of MTT solution (1 mg/ml) and then incubated for 4 hours. After

incubated, the MTT solution was removed and replaced with 100 µl of DMSO to dissolve the formed purple

formazan crystals. Each experiment was performed in triplicate. The absorbance was detected using a

spectrophotometer microplate reader (UV scan) at wavelength 570 nm. The absorption values of the solution

directly represented relative the surviving cells number. The percentage of cell viability was calculated in

accordance with the following equation: Cell viability (%) = [(absorbance of treated cells / absorbance of

untreated cells)] x 100. The results were shown in graph between the percentage of cell viability and

concentrations.

Thai Journal of Pharmaceutical Sciences (TJPS) The JSPS-NRCT Follow-Up Seminar 2017 and

33rd International Annual Meeting in Pharmaceutical Sciences

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TJPS 2017, 41 (Supplement Issue): 218

The mRNA expression of inflammatory mediator measurement: The effects of Thai rice bran extracts on

inflammatory mediator production, the mRNA expression of inflammatory mediators were determined by RT-

qPCR technique. L929 cells and RAW 264.7 cells were pre-treated or untreated with Thai rice bran extracts

(0.5 mg/ml) followed by LPS (10 µg/ml) stimulation. The mRNA from the cultured cells was extracted by using

Thermo Scientific GeneJET RNA Purification Kit. The expression of mRNA was detected by using KAPA

SYBR FAST One-step RT-qPCR kits and analyzed by Mx 3005p Real Time PCR system following the

manufacturer’s instructions. The gene specific primers for RT-qPCR (mouse) were designed and synthesized

as follow; COX-2 (sense, 5′-tgcatgtggctgtggatgtcatcaa-3′; antisense, 5′-cactaagacagacccgtcatctcca-3′),

GAPDH (sense, 5′-gcctgcttcaccaccttc-3′; antisense, 5′-ggctctccagaacatcatcc-3′), IL-6 (sense, 5′-

ccggagaggagacttcacag-3′; antisense, 5′-ggaaattggggtaggaagga-3′), IL-10 (sense, 5′-gctggacaacatactgctaacc-

3′; antisense, 5′-atttccgataaggcttggcaa-3′), iNOS (sense, 5′-gtgttccaccaggagatgttg-3′; antisense, 5′-

ctcctgcccactgagttcgtc-3′), and TNF-α (sense, 5′-tactgaacttcggggtgattggtcc-3′; antisense, 5′-

cagccttgtcccttgaagagaacc-3′)

Measurement of inflammatory mediator secretion: The inflammatory mediator, PGE2 and TNF-α, secretion

were determined by ELISA assay. L929 cells and RAW 264.7 cells were pre-treated in the presence or

absence of Thai rice bran extracts (0.5 mg/ml) before LPS (20 µg/ml) stimulation. After incubation, the

medium was collected for measurement of inflammatory mediator secretion, which were performed by using

PGE2 (Abcam, R&D) and TNF-α (Abcam) ELISA kit following the manufacturer’s instructions.

Results and Discussion We first performed MTT assay to evaluate cell toxicity and optimal concentration of the bran extracts

from Red Hawm, Red Rose, and Black Sukhothai II before studying anti-inflammatory activities of these three

Thai rice bran extracts in LPS-stimulated L929 and RAW 264.7 cells. The tested extracts were diluted 2

alternated with 5 fold (0.01-5 mg/ml) as the method previously described4 and by then run for MTT assay. We

found that Red Hawm, Red Rose, and Black Sukhothai II extracts with concentrations at 0.01-1 mg/ml did not

toxic to L929 and RAW 264.7 cells. Thus, we chose the Thai rice bran extracts at a concentration of 0.5 mg/ml

for further investigation. We next investigated whether Thai rice bran extracts inhibits LPS-induced

inflammatory mediator mRNA expression in L929 and RAW 264.7 cells. The mRNA expressions were

measured by using RT-qPCR. After incubation with LPS, the mRNA expression of COX-2, IL-6, IL-10, iNOS,

and TNF-α in L929 and RAW 264.7 cells (Figure1 and 2).

Figure 1. The effects of Thai rice bran extracts on mRNA expression of inflammatory mediators in LPS-induced L929 cells. L929 cells

were pre-treated with Red Hawm, Red Rose, and Black Sukhothai II extracts for 1 hour before stimulated by 10 µg/ml LPS for 12 hours at

37º C. After treatment, the total RNA was extracted and the mRNA expression of inflammatory mediators was analyzed by using specific

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TJPS 2017, 41 (Supplement Issue): 219

primers. The relative COX-2, IL-6, IL-10, iNOS, and TNF-α mRNA levels were quantified and shown as the mean ± SEM (n=3). #P<0.05

vs. control; *P<0.05 vs. LPS.

Figure 2. The effects of Thai rice bran extracts on mRNA expression of inflammatory mediators in LPS-induced RAW 264.7 cells. RAW

264.7 cells were pre-treated with Red Hawm, Red Rose, and Black Sukhothai II extracts for 1 hour before stimulated by 10 µg/ml LPS for

6 hours at 37º C. After treatment, the total RNA was extracted from the cells and the mRNA expression of inflammatory mediators was

analyzed by using specific primers. The relative COX-2, IL-6, IL-10, iNOS, and TNF-α mRNA levels were quantified and shown as the

mean ± SEM (n=3). #P<0.05 vs. control; *P<0.05 vs. LPS.

Red Hawm, Red Rose, and Black Sukhothai II rice are colored or pigmented Thai rice. Several

studies have provided evidence that colored rice extract and its active compounds exhibited anti-inflammatory

activities by suppressing the production of inflammatory mediators such as NO, PGE2, iNOS, COX-2, TNF-α,

IL-1β, and IL-6 in LPS-stimulated RAW 264.7 cells5,6. Nevertheless, the studies have not yet considered the

anti-inflammatory effects of Thai rice bran in inflammatory cells related to skin inflammation. As shown in

Figure 1 and 2, treatment with Red Hawm and Red Rose (0.5 mg/ml) significantly decreased mRNA

expression of COX-2, iNOS, and TNF-α in both L929 and RAW 264.7 cells and also significantly reduced

mRNA expression of IL-6 and IL-10 in L929 cells. Thus, the extracts of Red Hawm and Red Rose had the

anti-inflammatory effects on inflammatory mediator production.

Figure 3. Effects of Thai rice bran extracts on LPS-induced PGE2 and TNF-α secretion in L929 and RAW 264.7 cells. L929 and RAW

264.7 cells were pretreated with Red Hawm, Red Rose, and Black Sukhothai II extracts (0.5 mg/ml) for 1 and 3 hour and then stimulated

with 20 µg/ml LPS for 12 and 24 hours, respectively, at 37 °C. After LPS stimulation, the supernatant (medium) was collected. The PGE2

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TJPS 2017, 41 (Supplement Issue): 220

and TNF-α secretion into medium was assessed by ELISA assay. The PGE2 and TNF-α levels were quantified using standard curve and

expressed as the mean SEM (n=3). #P<0.05 vs. control; *P <0.05 vs. LPS.

We next investigated the anti-inflammatory property of Thai rice bran extracts on LPS-induced PGE2

and TNF-α secretion in L929 and RAW 264.7 cells (Figure 3). As shown in Figure 3, treatment with LPS (20

µg/ml) markedly increased PGE2 and TNF-α secretion as compared with control group. Pretreatment with

Thai rice bran extracts (0.5 mg/ml) did not inhibit the PGE2 and TNF-α secretion whereas dexamethasone

significantly decrease the LPS-induced PGE2 and TNF-α in RAW 264.7 cells and only PGE2 secretion in L929

cells. In contrast, our results were not the same as the studies by others in which TNF-α and PGE2 secretions

were not decreased after treatment with rice bran extracts in L929 and RAW 264.76,7.

Conclusion The extracts of Red Hawm and Red Rose had the anti-inflammatory effects. Our results supported the

hypothesis that the extracts of Red Hawm and Red Rose exerted anti-inflammatory effects by inhibiting the

LPS-stimulated production of COX-2, iNOS, and TNF-α in L929 and RAW 264.7 cells, but not the secretion of

PGE2 and TNF-α. Moreover, these extracts also suppressed the production of IL-6 and IL-10 in LPS-induced

L929 cells. Thus, the results from our study suggested that Red Hawm and Red Rose extracts may be a

potent anti-inflammatory therapeutic agent in skin-inflammatory diseases and skin aging.

Acknowledgements

This research was funded by the Thailand Research Fund (TRF) for Research and Researcher for

Industry Program (MSD57I0048) and S&J International Enterprises Public Company Limited. The authors also

gratefully thank Faculty of Pharmacy, Mahidol University for their facility supports.

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