Research ArticleIn Vitro Anti-Inflammatory Activity ofMorus alba L StemExtract in LPS-Stimulated RAW 2647 Cells

Nattaporn Soonthornsit1 Chetsadaporn Pitaksutheepong2 Warinkarn Hemstapat3

Pongsak Utaisincharoen4 and Tasana Pitaksuteepong1

1Department of Pharmaceutical Technology Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in ChemistryNaresuan University Tha Pho Mueang Phitsanulok Phitsanulok 65000 Thailand2Food Biotechnology Research Unit National Center for Genetic Engineering and Biotechnology (BIOTEC) 113Thailand Science ParkPhahonyothin Road Khlong Nueng Khlong Luang PathumThani 12120 Thailand3Department of Pharmacology Faculty of Science Mahidol University Rama VI Road Ratchathewi Bangkok 10400 Thailand4Department of Microbiology Faculty of Science Mahidol University Rama VI Road Ratchathewi Bangkok 10400 Thailand

Correspondence should be addressed to Tasana Pitaksuteepong tasanapnuacth

Received 8 February 2017 Revised 9 April 2017 Accepted 16 May 2017 Published 8 June 2017

Academic Editor Cheorl-Ho Kim

Copyright copy 2017 Nattaporn Soonthornsit et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Morus alba L also known as white mulberry or Mhon has long been used in traditional medicines This study was aimed toinvestigate anti-inflammatory activities of mulberry stem ethanolic extract (MSE) in lipopolysaccharide- (LPS-) stimulated RAW2647 macrophage cell line The MSE was first prepared and then investigated for cell viability using the MTT assay The anti-inflammatory activities were investigated through the inhibition of inducible nitric oxide synthase (iNOS) cyclooxygenase- (COX-)2 mRNA expression and iNOS protein expression using reverse transcription-polymerase chain reaction (RT-PCR) assay andimmunoblotting analysis respectively The inhibition of nitric oxide production of the MSE was also investigated using the Griessreaction assayTheMSE concentration ranging from 10 to 40 120583gml yielded cell viability higher than 80TheMSEat concentrationsof 20 and 40 120583gml demonstrated anti-inflammatory activity through the inhibition of nitric oxide production via suppression ofboth the iNOS mRNA and protein It was also found to inhibit the expression of COX-2 mRNA in LPS-induced RAW 2647 cellsThis study is the first to report the anti-inflammatory potential of the extract prepared from the stem of mulberry

1 Introduction

Morus alba (Moraceae) is known as mulberry or Mhonin Thai Its leaves fruit and bark have long been used intraditional Chinese medicine to treat fever improve eyesightstrengthen joints and lower blood pressure [1] The leavesare the most widely used and are consumed as an antihyper-glycemic supplement in Korea and Japan [1] Cultivation ofmulberry trees in Thailand is also widespread particularlyfor the leaves to feed silkworms The twigs and stems areoften pruned cut as normal cultivation practice and usedfor firewood but are rarely used for other purposes such asmedicinal use

A natural chemical compound found in the twigs andstems of mulberry trees is oxyresveratrol or trans-2 31015840 451015840-tetrahydroxystilbene This compound has been reported

to possess antioxidative and radical scavenging activitieswith IC50 value of 36 plusmn 00 120583M and 151 plusmn 23 120583M usingFeSO4H2O2-induced lipid peroxidation in rat liver micro-somes and DPPH assay respectively [2] It has also beenreported to have anti-inflammatory activity by inhibitingthe production of nitrite prostaglandin E2 and induciblenitric oxide synthase (iNOS) expression in the LPS-activatedRAW 2647 macrophage cells through the NF-120581B activationpathway [2] In addition oxyresveratrol has been shownto inhibit the rat paw edema induced by carrageenan in adose-dependent manner [2] Apart from the NF-120581B signalingpathway the anti-inflammatory effect of oxyresveratrol inLPS-induced RAW2647 cells has recently been reportedas also occurring through the inhibition of the mitogen-activated protein kinase (MAPK) pathway [3]

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 3928956 8 pageshttpsdoiorg10115520173928956

2 Evidence-Based Complementary and Alternative Medicine

Our previous study [4] found different amounts ofoxyresveratrol in various parts of the mulberry tree and theethanolic extract obtained from the mulberry stems has thehighest amount of oxyresveratrol compared to that obtainedfrom the twigs with the least amount in the leavesThereforewe hypothesized that the stem wood of mulberry treeswhich was left unused would also have anti-inflammatoryactivity

Inflammation is the bodyrsquos protective response thatintended to eliminate the initial cause of cell injury Howeversometimes it can also become self-perpetuating and causefurther inflammation Chronic inflammation is involved inmany diseases for example ulcerative colitis Alzheimerrsquosdermatitis cardiovascular diseases cancer inflammatorybowel disease (IBD) systemic lupus erythematous (SLE)osteoarthritis (OA) and rheumatoid arthritis (RA) [5] Oncemacrophages are elicited inflammatory mediators includingnitric oxide and proinflammatory mediators such as iNOSand COX-2 are produced There are few reports [1 6] onthe anti-inflammatory effect of the stems of mulberry treesTherefore this study was aimed to investigate the anti-inflammatory activities of mulberry stem extract (the MSE)through the inhibition of iNOS and COX-2 expression andNO production in LPS-stimulated RAW 2647 macrophagecells

2 Materials and Methods

21 Materials Oxyresveratrol isolated and purified fromArtocarpus lakoocha heartwood or Puag-Haad was preparedin-house using the method as previously described [7]HPLC grade acetonitrile was purchased from RCI Lab-scan (Bangkok Thailand) A mouse macrophage cell line(RAW 2647) was obtained from the American Type Cul-ture Collection (ATCC TIB-71 Manassas Virginia USA)Lipopolysaccharide (LPS) 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT) and dimethylsulfox-ide (DMSO) were purchased from Sigma-Aldrich (SaintLouis Missouri USA) RNAspin Mini RNA Isolation Kitwas purchased from GE Healthcare (Buckinghamshire UK)iScript cDNA Synthesis Kit was purchased from Bio-RadLaboratories (Hercules California USA) dNTPs were pur-chased from Fermentas (Vilnius Lithuania) Taq DNA poly-merase was purchased from Invitrogen (California USA)Griess Reagent Kit was purchased from Molecular ProbesInc (Eugene Oregon USA) Dulbeccorsquos modified Eaglersquosmedium (DMEM) and fetal bovine serum (FBS) were pur-chased fromHyClone (Logan Utah USA) L-Glutamine waspurchased from Biological Industries (Kibbutz Beit HaemekIsrael) Blocking solution was purchased from Roche Diag-nostics (Mannheim Germany) Specific monoclonal rabbitantibody to mouse iNOS was purchased from Santa CruzBiotechnology (Santa Cruz California USA) Specific mon-oclonal mouse antibody to 120573-actin was purchased from RampDsystems (Minnesota USA) Horseradish peroxidase- (HRP-)conjugated goat anti-rabbit IgG and HRP-conjugated rabbitanti-mouse IgG were purchased from Pierce (RockfordIllinois USA)

22 Preparation of the MSE Fresh mulberry stems (varBuriram 60) were supplied by the Queen Sirikit SericultureCenter Tak Province Thailand A voucher specimen wasverified byDr PraneeNangngamanddeposited at the Facultyof Science Naresuan University (voucher specimen number004067)The chopped and dried inner wood of the mulberrystems (3700 g) was macerated in 4 L of 80 ethanol Afterfiltration the filtrate was evaporated under reduced pressureusing a rotary evaporator (Buchi Rotavapor R-114 FlawilSwitzerland) and continued drying using a water bath (M25Lauda Deutschland Germany) The percentage yield of theextract was calculated using the following equation

yield = ( Dried weight of crude extractDried weight of chopped-dried plant

)times 100

(1)

23 High Performance Liquid Chromatography (HPLC) Anal-ysis of Oxyresveratrol in the MSE Oxyresveratrol was used asthe marker compound for the quantitative HPLC analysis ofMSE using the method described by Yhirayha [7] Standardstock solution 1mgml of oxyresveratrol was prepared by dis-solving in 80 ethanolThe stock solutionwas further dilutedto 5 standardworking solutions over the concentrations rangeof 00005 to 005mgml using 80 ethanol AnHPLC system(LC-20AT Shimadzu Kyoto Japan) equipped with a UV-Vis detector (SPD-20A Shimadzu Japan) an autosampler(SIL-10ADVP Shimadzu Kyoto Japan) and a column ovenwas used Analysis was performed on a C18 boned-silica gelcolumn (Gemini 5 u 150 times 46mm Phenomenex TorranceUSA) in the isocratic mode Acetonitrile mixed with 005Mphosphate buffer pH 3 (13 ratio) was used as the mobilephase at a flow rate of 1mlmin UV detector wavelength of320 nm with column oven temperature of 30∘C was set Theinjection volume was 20 120583l Run time was set at 13min Thestock solution of the extract was prepared in 80 ethanol atconcentration of 025mgml

24 Thin Layer Chromatography (TLC) Analysis The stocksolutions of the crude extract and oxyresveratrol were pre-pared by dissolving in ethanol at concentration 6mgmland 1mgml respectively Ascending TLC analysis was per-formed using silica gel 60 F254 (Merck Darmstadt Germany)as a stationary phase andmethanol chloroform (15 85 vv)as a mobile phase Small amount of samples was spotted atabout 05 cm from the bottom of the TLC plate and thenplaced in the chamber which was left to saturate with solventvapor for 15minThe developing time was allowed for 5minAfter that the plate was removed from the chamber andair-dried The TLC spots were viewed under UV light at254 and 366 nm Then anisaldehyde reagent was sprayed onTLC plate and then heated continuously to 100∘C in orderto visualize oxyresveratrol The colored spots appeared weremarked and the retention factor (119877119891) was calculated from thefollowing equation

119877119891 = distance traveled by a compounddistance traveled by solvent front

(2)

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The primer sequences used for PCR amplification

Gene Primer sequence (51015840-31015840) Product size (bp) PCR condition Reference

iNOS GCA GAA TGT GAC CAT CAT GGACA ACC TTG GTG TTG AAG GC 414

94∘C 5min

94∘C 45 sec60∘C 1min

72∘C 1min

]]]]]

30 cycles [8]

COX-2 AGA AGG AAA TGG CTG CAG AAGCT CGG CTT CCA GTA TTG AG 194

94∘C 5min

94∘C 30 sec54∘C 30 sec72∘C 30 sec

]]]]]

30 cycles This study

120573-Actin CCA GAG CAA GAG AGG TAT CCCTG TGG TGG TGA AGC TGT AG 436

94∘C 5min

94∘C 45 sec58∘C 1min

72∘C 1min

]]]]]

30 cycles [8]

25 Anti-Inflammatory Activity of the MSE

251 Sample Preparation The dried crude extract was dis-solved in 05 DMSO to make a final concentration of1mgml and then filtered through 022 120583m nylon membrane(VertiClean Vertical Chromatography Co Ltd BangkokThailand) before further analysis Oxyresveratrol a biologicalmarker in this study was also prepared in the same manneras the extract

252 Cell Culture and Viability The RAW 2647 cells werecultured in DMEM supplemented with 10 FBS and 1 L-glutamine and incubated at 37∘C in 5 CO2 To determinecell viability RAW 2647 cells were seeded in a 96-well plateat a density of 3 times 104 cellswell After overnight incubationthe cells were treated with various concentrations of samplesin the presence of 100 ngml LPS and incubated for 24 h Cellviability was determined by MTT assay

253 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay RAW 2647 cells were seeded in a 6-well plateat a concentration of 25 times 105 cellsml After overnightincubation the cells were resuspended in 1ml of supple-mented DMEM and pretreated with various concentrationsof samples for 2 h LPS 100 ngml was then added andfurther incubated for 6 h The cells were then washed twicewith 1ml PBS harvested in 1ml PBS and subjected toRT-PCR analysis Total RNA from the stimulated cells wasextracted using RNAspin Mini RNA Isolation Kit accordingto the manufacturerrsquos instruction cDNA was synthesizedusing iScript cDNA Synthesis Kit The TGradient thermalcycler 96 (Biometra Goettingen Germany) was used forPCR amplificationThe PCRmixture contained 50 ng cDNA1x PCR buffer 125mM MgCl2 100 120583M dNTPs 450 nM ofeach primer and 1U Taq polymerase The primers and PCRconditions used for amplification of iNOS COX-2 and 120573-actin genes are shown in Table 1The amplified products were

separated on 15 (wv) agarose gel After gel electrophoresiswas completed the gels were stained with ethidium bromideand the PCR products were visualized under anUV illumina-tor (GeneSys Rochester New York USA)The band intensityof iNOS and COX-2 genes was then normalized to 120573-actinand calculated as the relative expression

254 Immunoblotting Analysis RAW2647 cells were seededin a 6-well plate at a concentration of 19 times 105 cellsmlAfter overnight incubation they were resuspended in 1mlof supplemented DMEM and pretreated with various con-centrations of samples for 2 h before LPS (100 ngml) wasadded After 16 h of incubation the cell supernatants werecollected and subjected to NO production analysis (seeSection 255) The cells were washed twice with PBS andresuspended in 1ml PBS Following centrifugation of thecell sample at 10000 rpm the cell pellet was lysed in lysisbuffer pH 68 The mixtures were sonicated on ice for1min and then heated for 5min in a dry bath incubator(Boekel Scientific Pennsylvania USA) The mixture wascentrifuged at 10000 rpm at 4∘C for 5min The cell lysateswere electrophoresed on 8 SDS-PAGE and then electro-transferred to a nitrocellulose membrane The membranewas blocked with 5 blocking solution (Roche DiagnosticsMannheim Germany) in PBS for 1 h before incubating at 4∘Covernight with specific monoclonal rabbit antibody tomouseiNOS and specific monoclonal mouse antibody to 120573-actinThe concentration of each antibody was used according tothe manufacturerrsquos recommendation The membranes werewashed 3 times with 01 Tween 20 in PBS for 15minfollowed by reacting with HRP-conjugated goat anti-rabbitIgG (for iNOS) and HRP-conjugated rabbit anti-mouse IgG(for 120573-actin) at room temperature for 1 h After that themembrane was washed 4 times (20min each wash) with 01Tween 20 in PBS Protein bands were detected by enhancedchemiluminescence as recommended by the manufacturer(Roche Diagnostics Mannheim Germany) after exposure

4 Evidence-Based Complementary and Alternative Medicine

to hyperfilm (GE Healthcare Buckinghamshire UK) TheiNOS immunoblot signals were compared with 120573-actin andcalculated as the relative protein expression

255 NO Production Assay As detailed above the nitriteconcentration in cell supernatant was measured as an indica-tor of NO production using the Griess Reagent Kit Briefly150 120583l of each supernatant was mixed with 20120583l of Griessreagent (1 sulfanilamide in 5 phosphoric and 01 naph-thylethylenediamine dihydrochloride in water) and 130 120583l ofdistilledwater in a 96-well plateThemixtures were incubatedat room temperature for 30min and then the absorbanceof each well was determined at 548 nm using a microplatereader The amount of nitrite in samples was back-calculatedfrom a sodium nitrite calibration curve (0ndash100 120583M)

26 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) Results were obtained from at leastthree independent experiments each performed in triplicateStatistical analysis was determined using one-way analysis ofvariance (ANOVA) followed by the Tukeyrsquos test for multiplecomparisons (GraphPad Prism 60 GraphPad Software IncSan Diego USA) 119875 values less than or equal to 005 wereconsidered significant

3 Results and Discussion

The leaves fruit and bark of mulberry have long been usedto treat various illnesses and to improve health functionsHowever few reports have investigated the health benefits ofthe stem wood of mulberry which is abundantly available inThailand The anti-inflammatory effects were the biologicalactivity particularly investigated in this study

31 HPLC Analysis of Oxyresveratrol in the MSE First theMSE was prepared Ethanolic extract was obtained as apowder which had a dark brown color with a percentageyield of 513 of the raw woody material The amount ofoxyresveratrol in the MSE was analyzed by HPLC Thecalculated amount of oxyresveratrol in the MSE was 1787 plusmn061 [9]

32 TLC Analysis TLC analysis was performed to conductquality control of natural herbal product containing complexmixtures of compounds like in the case of mulberry stemextract Oxyresveratrol one of the bioactive compoundsfound in mulberry stem extract was used as a markerObserving under UV light at 254 nm and 366 nm the resultsshowed that mulberry stem extract showed eight spots with119877119891 values 002 009 016 044 064 071 078 and 087 whileoxyresveratrol showed only clear one spot with 119877119891 value 044by comparing 119877119891 value It was clearly shown that a reddishspot observed in mulberry stem extract after spraying TLCplate with anisaldehyde reagent with 119877119891 value of 044 wasoxyresveratrol (Figure 1)

33 Cell Viability To ensure that the cells were healthy beforeperforming the bioactivity assays and the tested concentra-tions were not toxic to the cells cell viability after treatment

Spraying with anisaldehyde reagent

Mulberry stem extractOxyresveratrol

UV light (휆 = 254 nm)UV light (휆 = 366 nm)

Figure 1 TLC fingerprint of mulberry stem extract Standardoxyresveratrol was used as a marker Mobile phase system wasmethanol chloroform (15 85 vv)

with various concentrations of the MSE was determinedThe concentrations of the MSE that yielded cell viabilityhigher than 80 were the main aim of this experiment TheRAW 2647 cell viability slightly decreased with increasingthe concentration of theMSE from 10 to 150120583gml (Figure 2)Identifying the concentrations of the MSE that yielded cellviability higher than 80 it was found to be in the range of10 to 40 120583gml and the concentrations of the MSE selectedfor subsequent studies were 20 and 40120583gml Oxyresveratrolwas included in this study as the marker of the MSE Anequivalent amount of oxyresveratrol in each concentrationof the extract tested (for example equivalent amount ofoxyresveratrol in the extract 100120583gml was 17 120583gml) wasalso evaluated for the effect on cell viability The resultswere shown to be similar to those observed in the MSE-treated cells (Figure 2) The anti-inflammatory effects ofoxyresveratrol were also confirmed in subsequent studies inwhich the selected concentrations of oxyresveratrol were 5and 10 120583gml

34 Anti-Inflammatory Activity of the MSE M alba extracthas been demonstrated to exhibit anti-inflammatory activitythrough the inhibition of iNOS NO and COX [10ndash15]However most of the extract tested was prepared from leaves[10 11 14] and root bark [12 13 15] In our study anti-inflammation activity of the extract prepared from mulberrystems was investigated

In an LPS-stimulated mouse macrophage RAW 2647cells model the NF-120581B signaling pathway plays a crucialrole in regulating inflammation through the transcriptionof iNOS COX and cytokine genes Found in the cyto-plasm of resting cells dimer NF-120581B is normally confinedto an inactive cytoplasmic complex through binding to aninhibitory protein I-120581B which masks its nuclear localizationsignal Exposure of cells to external proinflammatory stimulisuch as mitogens inflammatory cytokines and LPS causesrapid I-120581B phosphorylation by I-120581B kinase (IKK) resultingin free and subsequent nuclear translocation of NF-120581B [16

Evidence-Based Complementary and Alternative Medicine 5

010

10

20

20

30

30

40

40

50

50

60708090

100

100 150

MSE Oxyresveratrol

ce

ll vi

abili

ty

17 17minus + + + + + + + + + + + + + + +

34 51 68 85 255

l(휇gml)LPS 100 ngml

Figure 2 The percentage viability of RAW 2647 cells which was untreated (negative control) treated with only 100 ngml LPS (positivecontrol) and treated with various concentrations of MSE and oxyresveratrol in the presence of 100 ngml LPS for 24 h The viability of cellswas determined by MTT assay The data were represented as means plusmn SD of three independent experiments

iNOS

minus +++++

(휇gml)20 40 105

MSE Oxyresveratrol

β-Actin

LPS 100 ngml

(a)

lowastlowast

Rela

tive e

xpre

ssio

nle

vels

of iN

OS

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

080

060

040

020

000LPS 100 ngml

(b)

Figure 3 The effects of MSE and oxyresveratrol on the expression of iNOS mRNA in RAW 2647 cells The untreated cells were used asa negative control and the cells treated with only 100 ngml LPS were served as a positive control (a) RT-PCR analysis for detection ofiNOS mRNA expression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml oroxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS mRNA compared to 120573-actin The data wererepresented as means plusmn SD of three independent experiments The means marked with lowast are significantly different (119875 lt 005) from that ofthe cells treated with only LPS

17] In the nucleus NF-120581B induces the transcription of alarge variety of target proinflammatory genes including iNOSand COX-2 iNOS then further facilitates the conversionof L-arginine to L-citrulline and a large amount of NOwhile COX-2 catalyzes the conversion of arachidonic acid toprostaglandins Prostaglandins (PGs) especially PGE2 andPGI2 cause vasodilation that contribute to the vascular signsof inflammation and potentiate edema formation [18 19]

341 Effect of the MSE on the Expression of iNOS mRNAand Protein The anti-inflammatory effects of the MSE at theconcentrations of 20 and 40120583gml were investigated throughthe inhibition on the expression of iNOS mRNA and itsprotein as well as the production of NO Oxyresveratrol(marker) at the concentrations of 5 and 10 120583gml was alsotested 120573-Actin was used as an internal standard for RT-PCRassay As expected the expression of iNOS mRNA and itsproteinwas detected by stimulatingRAW2647 cells with LPS(Figures 3 and 4)The expression level of iNOS in cells treatedwith theMSE at the concentration of 40 120583gml was decreased

by 50 compared with that in cells treated with LPS alone(Figure 3) Similar results were observed in cells treated withoxyresveratrol

Western blotting was further performed to confirm theeffects of the MSE on iNOS protein expression The resultsshowed that iNOS protein was significantly decreased inuntreated RAW 2647 cells comparing with LPS-treatedRAW 2647 cells (Figure 4) Therefore the validity of theiNOS protein assay was again confirmed The MSE at theconcentrations of 20 (P lt 005) and 40 (P lt 0001)120583gml andoxyresveratrol at 10 (P lt 005) 120583gml significantly inhibitedthe expression of iNOS protein compared with the cellstreated only with LPS

342 Effect of the MSE on NO Production The inducibleforms of NOS are the most important proinflammatoryenzymes responsible for increasing the levels of NO There-fore the effect of tested compounds on the inhibition ofNO production was further investigated However NO inthe biological matrix is very unstable and rapidly oxidizes

6 Evidence-Based Complementary and Alternative Medicine

MSE Oxyresveratrol

minus +++++

20 40 10 (휇gml)5

iNOS

β-Actin

LPS 100 ngml

(a)

000

020

040

060

080

100

MSE

lowast lowast

lowastlowastlowast

Rela

tive e

xpre

ssio

n le

vels

of iN

OS

prot

ein

minus +++++

20 40 10 (휇gml)5

Oxyresveratrol

LPS 100 ngml

(b)

Figure 4 The effects of MSE and oxyresveratrol on the expression of iNOS protein in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) Immunoblotting analysis for determinationthe expression of iNOS protein in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS protein compared to 120573-actin The datawere represented as means plusmn SD of three independent experiments The means marked with lowast lowastlowastlowast are significantly different (119875 lt 005) and(119875 lt 0001) respectively from that of the cells treated with only LPS

Nitr

ite (휇

M)

minus +++++

20 40 10 (휇gml)5

MSE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Oxyresveratrol

2500

2000

1500

1000

500

000

LPS 100 ngml

Figure 5 The effects of MSE and oxyresveratrol on the productionof nitrite in LPS-stimulated RAW 2647 cells which were untreated(negative control) treated with only 100 ngml LPS (positive con-trol) pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations of 5 and 10120583gml for 2 hand then treated with 100 ngml LPS for 16 h The supernatant wascollected and determined by Griess reaction assay The data wererepresented as means plusmn SD of three independent experiments Themeansmarkedwithlowastlowastlowastlowastlowastlowast are significantly different (119875 lt 005)(119875 lt 0005) and (119875 lt 0001) respectively from that of the cellstreated with only LPS

to nitrite (NO2minus) and thus the measurement of nitrite is

routinely used as an index of NO productionFor the untreated RAW 2647 cells the concentration

of nitrite could not be detected (Figure 5) Once the cellswere stimulated with LPS a high concentration of nitrite wasproduced The MSE at concentrations of 20 and 40 120583gmlwere shown to inhibit nitrite production by 34 and 54respectively and oxyresveratrol at concentrations of 5 and10 120583gml was able to inhibit nitrite production by 16 and28 respectively (Figure 5)

343 Effect of the MSE on COX-2 mRNA Expression Asshown in Figure 6 theMSE was able to inhibit the expression

of COX-2 mRNA in LPS-activated RAW 2647 cells Theinhibitory activity of the MSE at 40 120583gml was shown to behigher than that of the MSE at 20 120583gmlThe expression levelof COX-2 mRNA in cells treated with the MSE at 40 120583gmldecreased by 70 compared with that in cells treated withLPS alone Similar results were observed when LPS-activatedRAW 2647 cells were incubated with oxyresveratrol

The results observed in this study clearly showed that theMSE could inhibit NO production through the inhibitionof both iNOS mRNA and protein expression In additionthe extract was shown to suppress the expression of COX-2 mRNA in LPS-stimulated RAW 2647 cells The inhibitioneffect on production of iNOS COX-2 and NO of the MSEat 40 120583gml was found to be higher than that of the MSE at20120583gml The MSE showed more potent inhibitory effectsthan oxyresveratrol at an equivalent amount of oxyresver-atrol (the MSE at 40 120583gml equivalent to 68120583gml) It hasbeen recently reported that the stems of M alba containedflavonoids (morusin) stilbenoids (mulberroside resveratroland oxyresveratrol) and coumarins [1] These polyphenolswere shown to possess anti-inflammatory effects [20ndash22]Wealso determined the amounts of total phenolic of the extractand it was 3487 plusmn 112120583mol GAEmg extract Thereforethese polyphenols other than oxyresveratrol may explainthe superior anti-inflammatory effect of the MSE Althoughthe precise molecular mechanisms responsible for the anti-inflammatory activities of theMSE cannot be explained in thepresent study oxyresveratrol which is an active componentpresented in the extract has been reported to exhibit its anti-inflammatory effect via the inhibition of the NF-120581B and theMAPK pathways [2 3] However further elucidation of theinsight mechanisms involved the anti-inflammatory effect ofthis plant extract is required

4 Conclusions

This study is the first to demonstrate the potential anti-inflammatory effects of the extract prepared from mulberry

Evidence-Based Complementary and Alternative Medicine 7

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

COX-2

β-Actin

LPS 100 ngml

(a)

000

030

060

090

120

150

Relat

ive e

xpre

ssio

n le

vels

of COX-2

minus +++++

20 40 10 (휇gml)5

lowastlowastlowast

lowastlowast lowastlowast

MSE Oxyresveratrol

LPS 100 ngml

(b)

Figure 6 The effects of MSE and oxyresveratrol on the expression of COX-2 mRNA in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) RT-PCR analysis for detection of COX-2mRNAexpression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml or oxyresveratrolat the concentrations of 5 and 10120583gml for 6 h (b) The relative expression of COX-2 mRNA compared to 120573-actin The data were representedas means plusmn SD of three independent experiments The means marked with lowastlowast lowastlowastlowast are significantly different (119875 lt 0005) and (119875 lt 0001)respectively from that of the cells treated with only LPS

stems on LPS-induced RAW 2647 cells The investigationfound that concentrations at 20 and 40 120583gml had no toxiceffect The results suggest that the stem extract mediates itsanti-inflammatory activities through the inhibition of iNOSNO and COX-2 production Further studies are necessary tofully define the precise mechanisms involved

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors would like to acknowledge the financial supportfrom the yearly budget of Naresuan University Thailand(R2555B069 and R2558B053) the Center for Innovation inChemistry (PERCH-CIC) and National Research Council ofThailand Graduate Student Scholarship (2014) They expressspecial gratitude towards theQueen Sirikit SericultureCenter(Tak) for the supply of plant materials and to Mr CheinYhirayha for the purification of the oxyresveratrol Manythanks are due toMr RoyMorien of the Naresuan UniversityLanguage Centre for his editing assistance and advice onEnglish expression in this manuscript

References

[1] E W-C Chan P-Y Lye and S-K Wong ldquoPhytochemistrypharmacology and clinical trials of Morus albardquo Chinese Jour-nal of Natural Medicines vol 14 no 1 pp 17ndash30 2016

[2] K-O Chung B-Y Kim M-H Lee et al ldquoIn-vitro and in-vivoanti-inflammatory effect of oxyresveratrol fromMorus alba LrdquoJournal of Pharmacy and Pharmacology vol 55 no 12 pp 1695ndash1700 2003

[3] H S Lee D H Kim J E Hong J-Y Lee and E JKim ldquoOxyresveratrol suppresses lipopolysaccharide-induced

inflammatory responses in murine macrophagesrdquo Human andExperimental Toxicology vol 34 no 8 pp 808ndash818 2015

[4] P Thongsuk In vitro and clinical study of mulberry extractfor skin whitening product [MS thesis] Naresuan UniversityPhitsanulok Thailand 2007

[5] D Laveti M Kumar and R Hemalatha ldquoAnti-inflammatorytreatments for chronic diseases a reviewrdquo Inflammation ampAllergymdashDrug Targets vol 12 no 5 pp 349ndash361 2013

[6] Y-C Chen Y-J Tien C-H Chen et al ldquoMorus alba and activecompound oxyresveratrol exert anti-inflammatory activity viainhibition of leukocyte migration involving MEKERK signal-ingrdquo BMCComplementary and AlternativeMedicine vol 13 no1 pp 45ndash55 2013

[7] C Yhirayha Formulation and skin penetration study of lyotropicliquid crystal incorporating mulberry stem (Morus alba L)extract [MS thesis] Naresuan University Phitsanulok Thai-land 2013

[8] M Pudla K Limposuwan and P Utaisincharoen ldquoBurkholde-ria pseudomallei-Induced expression of a negative regulatorsterile-120572 and Armadillo motif-containing protein in mousemacrophages a possible mechanism for suppression of theMyD88-independent pathwayrdquo Infection and Immunity vol 79no 7 pp 2921ndash2927 2011

[9] N Soonthornsit and T Pitaksuteepong ldquoMicroemulsion for-mulation containing mulberry stem extractrdquo in Proceedings ofthe 38th Congress on Science and Technology of Thailand 6pages Chiang Mai Thailand October 2012

[10] C H Hong S K Hur O-J Oh S S Kim K A Nam and S KLee ldquoEvaluation of natural products on inhibition of induciblecyclooxygenase (COX-2) and nitric oxide synthase (iNOS) incultured mouse macrophage cellsrdquo Journal of Ethnopharmacol-ogy vol 83 no 1-2 pp 153ndash159 2002

[11] E-M Choi and J-K Hwang ldquoEffects of Morus alba leafextract on the production of nitric oxide prostaglandin E2 andcytokines in RAW2647 macrophagesrdquo Fitoterapia vol 76 no7-8 pp 608ndash613 2005

[12] Z-G Yang K Matsuzaki S Takamatsu and S KitanakaldquoInhibitory effects of constituents from Morus alba var mul-ticaulis on Differentiation of 3T3-L1 cells and nitric Oxide

8 Evidence-Based Complementary and Alternative Medicine

Production in RAW2647 Cellsrdquo Molecules vol 16 no 7 pp6010ndash6022 2011

[13] H J Lim H-G Jin E-R Woo S K Lee and H P Kim ldquoTheroot barks ofMorus alba and the flavonoid constituents inhibitairway inflammationrdquo Journal of Ethnopharmacology vol 149no 1 pp 169ndash175 2013

[14] E Park S-M Lee J E Lee and J-H Kim ldquoAnti-inflammatoryactivity of mulberry leaf extract through inhibition of NF-120581BrdquoJournal of Functional Foods vol 5 no 1 pp 178ndash186 2013

[15] H Zelova Z Hanakova Z Cermakova et al ldquoEvaluation ofanti-inflammatory activity of prenylated substances isolatedfromMorus alba andMorus nigrardquo Journal of Natural Productsvol 77 no 6 pp 1297ndash1303 2014

[16] A Abate S Oberle and H Schroder ldquoLipopolysaccharide-induced expression of cyclooxygenase-2 inmousemacrophagesis inhibited by chloromethylketones and a direct inhibitor ofNF-120581B translocationrdquo Prostaglandins and Other Lipid Media-tors vol 56 no 5-6 pp 277ndash290 1998

[17] S-H Lee C-HKwak S-K Lee et al ldquoAnti-inflammatory effectof ascochlorin in lps-stimulated raw 2647 macrophage cells isaccompanied with the down-regulation of iNOS COX-2 andproinflammatory cytokines through NF-120581B ERK12 and p38signaling pathwayrdquo Journal of Cellular Biochemistry vol 117 no4 pp 978ndash987 2016

[18] L Parente ldquoPros and Cons of Selective Inhibition of COX-2versus Dual LipoxygenaseCOX Inhibition Is Two Better thanOnerdquo The Journal of Rheumatology vol 28 no 11 pp 2375ndash2382 2001

[19] U N Das ldquoCyclooxygenase (COX) lipoxygenase (LO) path-ways and generation of lipoxins resolvins protections andmaresinsrdquo in Molecular Basis of Health and Disease N DUndurti Ed vol 4 pp 59ndash61 Springer Science BusinessMediaBV Dordrecht Holland 2010

[20] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[21] C Riviere S Krisa L Pechamat et al ldquoPolyphenols from thestems of Morus alba and their inhibitory activity against nitricoxide production by lipopolysaccharide-activated microgliardquoFitoterapia vol 97 pp 253ndash260 2014

[22] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation and Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The primer sequences used for PCR amplification

Gene Primer sequence (51015840-31015840) Product size (bp) PCR condition Reference

iNOS GCA GAA TGT GAC CAT CAT GGACA ACC TTG GTG TTG AAG GC 414

94∘C 5min

94∘C 45 sec60∘C 1min

72∘C 1min

]]]]]

30 cycles [8]

COX-2 AGA AGG AAA TGG CTG CAG AAGCT CGG CTT CCA GTA TTG AG 194

94∘C 5min

94∘C 30 sec54∘C 30 sec72∘C 30 sec

]]]]]

30 cycles This study

120573-Actin CCA GAG CAA GAG AGG TAT CCCTG TGG TGG TGA AGC TGT AG 436

94∘C 5min

94∘C 45 sec58∘C 1min

72∘C 1min

]]]]]

30 cycles [8]

25 Anti-Inflammatory Activity of the MSE

251 Sample Preparation The dried crude extract was dis-solved in 05 DMSO to make a final concentration of1mgml and then filtered through 022 120583m nylon membrane(VertiClean Vertical Chromatography Co Ltd BangkokThailand) before further analysis Oxyresveratrol a biologicalmarker in this study was also prepared in the same manneras the extract

252 Cell Culture and Viability The RAW 2647 cells werecultured in DMEM supplemented with 10 FBS and 1 L-glutamine and incubated at 37∘C in 5 CO2 To determinecell viability RAW 2647 cells were seeded in a 96-well plateat a density of 3 times 104 cellswell After overnight incubationthe cells were treated with various concentrations of samplesin the presence of 100 ngml LPS and incubated for 24 h Cellviability was determined by MTT assay

253 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay RAW 2647 cells were seeded in a 6-well plateat a concentration of 25 times 105 cellsml After overnightincubation the cells were resuspended in 1ml of supple-mented DMEM and pretreated with various concentrationsof samples for 2 h LPS 100 ngml was then added andfurther incubated for 6 h The cells were then washed twicewith 1ml PBS harvested in 1ml PBS and subjected toRT-PCR analysis Total RNA from the stimulated cells wasextracted using RNAspin Mini RNA Isolation Kit accordingto the manufacturerrsquos instruction cDNA was synthesizedusing iScript cDNA Synthesis Kit The TGradient thermalcycler 96 (Biometra Goettingen Germany) was used forPCR amplificationThe PCRmixture contained 50 ng cDNA1x PCR buffer 125mM MgCl2 100 120583M dNTPs 450 nM ofeach primer and 1U Taq polymerase The primers and PCRconditions used for amplification of iNOS COX-2 and 120573-actin genes are shown in Table 1The amplified products were

separated on 15 (wv) agarose gel After gel electrophoresiswas completed the gels were stained with ethidium bromideand the PCR products were visualized under anUV illumina-tor (GeneSys Rochester New York USA)The band intensityof iNOS and COX-2 genes was then normalized to 120573-actinand calculated as the relative expression

254 Immunoblotting Analysis RAW2647 cells were seededin a 6-well plate at a concentration of 19 times 105 cellsmlAfter overnight incubation they were resuspended in 1mlof supplemented DMEM and pretreated with various con-centrations of samples for 2 h before LPS (100 ngml) wasadded After 16 h of incubation the cell supernatants werecollected and subjected to NO production analysis (seeSection 255) The cells were washed twice with PBS andresuspended in 1ml PBS Following centrifugation of thecell sample at 10000 rpm the cell pellet was lysed in lysisbuffer pH 68 The mixtures were sonicated on ice for1min and then heated for 5min in a dry bath incubator(Boekel Scientific Pennsylvania USA) The mixture wascentrifuged at 10000 rpm at 4∘C for 5min The cell lysateswere electrophoresed on 8 SDS-PAGE and then electro-transferred to a nitrocellulose membrane The membranewas blocked with 5 blocking solution (Roche DiagnosticsMannheim Germany) in PBS for 1 h before incubating at 4∘Covernight with specific monoclonal rabbit antibody tomouseiNOS and specific monoclonal mouse antibody to 120573-actinThe concentration of each antibody was used according tothe manufacturerrsquos recommendation The membranes werewashed 3 times with 01 Tween 20 in PBS for 15minfollowed by reacting with HRP-conjugated goat anti-rabbitIgG (for iNOS) and HRP-conjugated rabbit anti-mouse IgG(for 120573-actin) at room temperature for 1 h After that themembrane was washed 4 times (20min each wash) with 01Tween 20 in PBS Protein bands were detected by enhancedchemiluminescence as recommended by the manufacturer(Roche Diagnostics Mannheim Germany) after exposure

4 Evidence-Based Complementary and Alternative Medicine

to hyperfilm (GE Healthcare Buckinghamshire UK) TheiNOS immunoblot signals were compared with 120573-actin andcalculated as the relative protein expression

255 NO Production Assay As detailed above the nitriteconcentration in cell supernatant was measured as an indica-tor of NO production using the Griess Reagent Kit Briefly150 120583l of each supernatant was mixed with 20120583l of Griessreagent (1 sulfanilamide in 5 phosphoric and 01 naph-thylethylenediamine dihydrochloride in water) and 130 120583l ofdistilledwater in a 96-well plateThemixtures were incubatedat room temperature for 30min and then the absorbanceof each well was determined at 548 nm using a microplatereader The amount of nitrite in samples was back-calculatedfrom a sodium nitrite calibration curve (0ndash100 120583M)

26 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) Results were obtained from at leastthree independent experiments each performed in triplicateStatistical analysis was determined using one-way analysis ofvariance (ANOVA) followed by the Tukeyrsquos test for multiplecomparisons (GraphPad Prism 60 GraphPad Software IncSan Diego USA) 119875 values less than or equal to 005 wereconsidered significant

3 Results and Discussion

The leaves fruit and bark of mulberry have long been usedto treat various illnesses and to improve health functionsHowever few reports have investigated the health benefits ofthe stem wood of mulberry which is abundantly available inThailand The anti-inflammatory effects were the biologicalactivity particularly investigated in this study

31 HPLC Analysis of Oxyresveratrol in the MSE First theMSE was prepared Ethanolic extract was obtained as apowder which had a dark brown color with a percentageyield of 513 of the raw woody material The amount ofoxyresveratrol in the MSE was analyzed by HPLC Thecalculated amount of oxyresveratrol in the MSE was 1787 plusmn061 [9]

32 TLC Analysis TLC analysis was performed to conductquality control of natural herbal product containing complexmixtures of compounds like in the case of mulberry stemextract Oxyresveratrol one of the bioactive compoundsfound in mulberry stem extract was used as a markerObserving under UV light at 254 nm and 366 nm the resultsshowed that mulberry stem extract showed eight spots with119877119891 values 002 009 016 044 064 071 078 and 087 whileoxyresveratrol showed only clear one spot with 119877119891 value 044by comparing 119877119891 value It was clearly shown that a reddishspot observed in mulberry stem extract after spraying TLCplate with anisaldehyde reagent with 119877119891 value of 044 wasoxyresveratrol (Figure 1)

33 Cell Viability To ensure that the cells were healthy beforeperforming the bioactivity assays and the tested concentra-tions were not toxic to the cells cell viability after treatment

Spraying with anisaldehyde reagent

Mulberry stem extractOxyresveratrol

UV light (휆 = 254 nm)UV light (휆 = 366 nm)

Figure 1 TLC fingerprint of mulberry stem extract Standardoxyresveratrol was used as a marker Mobile phase system wasmethanol chloroform (15 85 vv)

with various concentrations of the MSE was determinedThe concentrations of the MSE that yielded cell viabilityhigher than 80 were the main aim of this experiment TheRAW 2647 cell viability slightly decreased with increasingthe concentration of theMSE from 10 to 150120583gml (Figure 2)Identifying the concentrations of the MSE that yielded cellviability higher than 80 it was found to be in the range of10 to 40 120583gml and the concentrations of the MSE selectedfor subsequent studies were 20 and 40120583gml Oxyresveratrolwas included in this study as the marker of the MSE Anequivalent amount of oxyresveratrol in each concentrationof the extract tested (for example equivalent amount ofoxyresveratrol in the extract 100120583gml was 17 120583gml) wasalso evaluated for the effect on cell viability The resultswere shown to be similar to those observed in the MSE-treated cells (Figure 2) The anti-inflammatory effects ofoxyresveratrol were also confirmed in subsequent studies inwhich the selected concentrations of oxyresveratrol were 5and 10 120583gml

34 Anti-Inflammatory Activity of the MSE M alba extracthas been demonstrated to exhibit anti-inflammatory activitythrough the inhibition of iNOS NO and COX [10ndash15]However most of the extract tested was prepared from leaves[10 11 14] and root bark [12 13 15] In our study anti-inflammation activity of the extract prepared from mulberrystems was investigated

In an LPS-stimulated mouse macrophage RAW 2647cells model the NF-120581B signaling pathway plays a crucialrole in regulating inflammation through the transcriptionof iNOS COX and cytokine genes Found in the cyto-plasm of resting cells dimer NF-120581B is normally confinedto an inactive cytoplasmic complex through binding to aninhibitory protein I-120581B which masks its nuclear localizationsignal Exposure of cells to external proinflammatory stimulisuch as mitogens inflammatory cytokines and LPS causesrapid I-120581B phosphorylation by I-120581B kinase (IKK) resultingin free and subsequent nuclear translocation of NF-120581B [16

Evidence-Based Complementary and Alternative Medicine 5

010

10

20

20

30

30

40

40

50

50

60708090

100

100 150

MSE Oxyresveratrol

ce

ll vi

abili

ty

17 17minus + + + + + + + + + + + + + + +

34 51 68 85 255

l(휇gml)LPS 100 ngml

Figure 2 The percentage viability of RAW 2647 cells which was untreated (negative control) treated with only 100 ngml LPS (positivecontrol) and treated with various concentrations of MSE and oxyresveratrol in the presence of 100 ngml LPS for 24 h The viability of cellswas determined by MTT assay The data were represented as means plusmn SD of three independent experiments

iNOS

minus +++++

(휇gml)20 40 105

MSE Oxyresveratrol

β-Actin

LPS 100 ngml

(a)

lowastlowast

Rela

tive e

xpre

ssio

nle

vels

of iN

OS

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

080

060

040

020

000LPS 100 ngml

(b)

Figure 3 The effects of MSE and oxyresveratrol on the expression of iNOS mRNA in RAW 2647 cells The untreated cells were used asa negative control and the cells treated with only 100 ngml LPS were served as a positive control (a) RT-PCR analysis for detection ofiNOS mRNA expression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml oroxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS mRNA compared to 120573-actin The data wererepresented as means plusmn SD of three independent experiments The means marked with lowast are significantly different (119875 lt 005) from that ofthe cells treated with only LPS

17] In the nucleus NF-120581B induces the transcription of alarge variety of target proinflammatory genes including iNOSand COX-2 iNOS then further facilitates the conversionof L-arginine to L-citrulline and a large amount of NOwhile COX-2 catalyzes the conversion of arachidonic acid toprostaglandins Prostaglandins (PGs) especially PGE2 andPGI2 cause vasodilation that contribute to the vascular signsof inflammation and potentiate edema formation [18 19]

341 Effect of the MSE on the Expression of iNOS mRNAand Protein The anti-inflammatory effects of the MSE at theconcentrations of 20 and 40120583gml were investigated throughthe inhibition on the expression of iNOS mRNA and itsprotein as well as the production of NO Oxyresveratrol(marker) at the concentrations of 5 and 10 120583gml was alsotested 120573-Actin was used as an internal standard for RT-PCRassay As expected the expression of iNOS mRNA and itsproteinwas detected by stimulatingRAW2647 cells with LPS(Figures 3 and 4)The expression level of iNOS in cells treatedwith theMSE at the concentration of 40 120583gml was decreased

by 50 compared with that in cells treated with LPS alone(Figure 3) Similar results were observed in cells treated withoxyresveratrol

Western blotting was further performed to confirm theeffects of the MSE on iNOS protein expression The resultsshowed that iNOS protein was significantly decreased inuntreated RAW 2647 cells comparing with LPS-treatedRAW 2647 cells (Figure 4) Therefore the validity of theiNOS protein assay was again confirmed The MSE at theconcentrations of 20 (P lt 005) and 40 (P lt 0001)120583gml andoxyresveratrol at 10 (P lt 005) 120583gml significantly inhibitedthe expression of iNOS protein compared with the cellstreated only with LPS

342 Effect of the MSE on NO Production The inducibleforms of NOS are the most important proinflammatoryenzymes responsible for increasing the levels of NO There-fore the effect of tested compounds on the inhibition ofNO production was further investigated However NO inthe biological matrix is very unstable and rapidly oxidizes

6 Evidence-Based Complementary and Alternative Medicine

MSE Oxyresveratrol

minus +++++

20 40 10 (휇gml)5

iNOS

β-Actin

LPS 100 ngml

(a)

000

020

040

060

080

100

MSE

lowast lowast

lowastlowastlowast

Rela

tive e

xpre

ssio

n le

vels

of iN

OS

prot

ein

minus +++++

20 40 10 (휇gml)5

Oxyresveratrol

LPS 100 ngml

(b)

Figure 4 The effects of MSE and oxyresveratrol on the expression of iNOS protein in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) Immunoblotting analysis for determinationthe expression of iNOS protein in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS protein compared to 120573-actin The datawere represented as means plusmn SD of three independent experiments The means marked with lowast lowastlowastlowast are significantly different (119875 lt 005) and(119875 lt 0001) respectively from that of the cells treated with only LPS

Nitr

ite (휇

M)

minus +++++

20 40 10 (휇gml)5

MSE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Oxyresveratrol

2500

2000

1500

1000

500

000

LPS 100 ngml

Figure 5 The effects of MSE and oxyresveratrol on the productionof nitrite in LPS-stimulated RAW 2647 cells which were untreated(negative control) treated with only 100 ngml LPS (positive con-trol) pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations of 5 and 10120583gml for 2 hand then treated with 100 ngml LPS for 16 h The supernatant wascollected and determined by Griess reaction assay The data wererepresented as means plusmn SD of three independent experiments Themeansmarkedwithlowastlowastlowastlowastlowastlowast are significantly different (119875 lt 005)(119875 lt 0005) and (119875 lt 0001) respectively from that of the cellstreated with only LPS

to nitrite (NO2minus) and thus the measurement of nitrite is

routinely used as an index of NO productionFor the untreated RAW 2647 cells the concentration

of nitrite could not be detected (Figure 5) Once the cellswere stimulated with LPS a high concentration of nitrite wasproduced The MSE at concentrations of 20 and 40 120583gmlwere shown to inhibit nitrite production by 34 and 54respectively and oxyresveratrol at concentrations of 5 and10 120583gml was able to inhibit nitrite production by 16 and28 respectively (Figure 5)

343 Effect of the MSE on COX-2 mRNA Expression Asshown in Figure 6 theMSE was able to inhibit the expression

of COX-2 mRNA in LPS-activated RAW 2647 cells Theinhibitory activity of the MSE at 40 120583gml was shown to behigher than that of the MSE at 20 120583gmlThe expression levelof COX-2 mRNA in cells treated with the MSE at 40 120583gmldecreased by 70 compared with that in cells treated withLPS alone Similar results were observed when LPS-activatedRAW 2647 cells were incubated with oxyresveratrol

The results observed in this study clearly showed that theMSE could inhibit NO production through the inhibitionof both iNOS mRNA and protein expression In additionthe extract was shown to suppress the expression of COX-2 mRNA in LPS-stimulated RAW 2647 cells The inhibitioneffect on production of iNOS COX-2 and NO of the MSEat 40 120583gml was found to be higher than that of the MSE at20120583gml The MSE showed more potent inhibitory effectsthan oxyresveratrol at an equivalent amount of oxyresver-atrol (the MSE at 40 120583gml equivalent to 68120583gml) It hasbeen recently reported that the stems of M alba containedflavonoids (morusin) stilbenoids (mulberroside resveratroland oxyresveratrol) and coumarins [1] These polyphenolswere shown to possess anti-inflammatory effects [20ndash22]Wealso determined the amounts of total phenolic of the extractand it was 3487 plusmn 112120583mol GAEmg extract Thereforethese polyphenols other than oxyresveratrol may explainthe superior anti-inflammatory effect of the MSE Althoughthe precise molecular mechanisms responsible for the anti-inflammatory activities of theMSE cannot be explained in thepresent study oxyresveratrol which is an active componentpresented in the extract has been reported to exhibit its anti-inflammatory effect via the inhibition of the NF-120581B and theMAPK pathways [2 3] However further elucidation of theinsight mechanisms involved the anti-inflammatory effect ofthis plant extract is required

4 Conclusions

This study is the first to demonstrate the potential anti-inflammatory effects of the extract prepared from mulberry

Evidence-Based Complementary and Alternative Medicine 7

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

COX-2

β-Actin

LPS 100 ngml

(a)

000

030

060

090

120

150

Relat

ive e

xpre

ssio

n le

vels

of COX-2

minus +++++

20 40 10 (휇gml)5

lowastlowastlowast

lowastlowast lowastlowast

MSE Oxyresveratrol

LPS 100 ngml

(b)

Figure 6 The effects of MSE and oxyresveratrol on the expression of COX-2 mRNA in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) RT-PCR analysis for detection of COX-2mRNAexpression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml or oxyresveratrolat the concentrations of 5 and 10120583gml for 6 h (b) The relative expression of COX-2 mRNA compared to 120573-actin The data were representedas means plusmn SD of three independent experiments The means marked with lowastlowast lowastlowastlowast are significantly different (119875 lt 0005) and (119875 lt 0001)respectively from that of the cells treated with only LPS

stems on LPS-induced RAW 2647 cells The investigationfound that concentrations at 20 and 40 120583gml had no toxiceffect The results suggest that the stem extract mediates itsanti-inflammatory activities through the inhibition of iNOSNO and COX-2 production Further studies are necessary tofully define the precise mechanisms involved

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors would like to acknowledge the financial supportfrom the yearly budget of Naresuan University Thailand(R2555B069 and R2558B053) the Center for Innovation inChemistry (PERCH-CIC) and National Research Council ofThailand Graduate Student Scholarship (2014) They expressspecial gratitude towards theQueen Sirikit SericultureCenter(Tak) for the supply of plant materials and to Mr CheinYhirayha for the purification of the oxyresveratrol Manythanks are due toMr RoyMorien of the Naresuan UniversityLanguage Centre for his editing assistance and advice onEnglish expression in this manuscript

References

[1] E W-C Chan P-Y Lye and S-K Wong ldquoPhytochemistrypharmacology and clinical trials of Morus albardquo Chinese Jour-nal of Natural Medicines vol 14 no 1 pp 17ndash30 2016

[2] K-O Chung B-Y Kim M-H Lee et al ldquoIn-vitro and in-vivoanti-inflammatory effect of oxyresveratrol fromMorus alba LrdquoJournal of Pharmacy and Pharmacology vol 55 no 12 pp 1695ndash1700 2003

[3] H S Lee D H Kim J E Hong J-Y Lee and E JKim ldquoOxyresveratrol suppresses lipopolysaccharide-induced

inflammatory responses in murine macrophagesrdquo Human andExperimental Toxicology vol 34 no 8 pp 808ndash818 2015

[4] P Thongsuk In vitro and clinical study of mulberry extractfor skin whitening product [MS thesis] Naresuan UniversityPhitsanulok Thailand 2007

[5] D Laveti M Kumar and R Hemalatha ldquoAnti-inflammatorytreatments for chronic diseases a reviewrdquo Inflammation ampAllergymdashDrug Targets vol 12 no 5 pp 349ndash361 2013

[6] Y-C Chen Y-J Tien C-H Chen et al ldquoMorus alba and activecompound oxyresveratrol exert anti-inflammatory activity viainhibition of leukocyte migration involving MEKERK signal-ingrdquo BMCComplementary and AlternativeMedicine vol 13 no1 pp 45ndash55 2013

[7] C Yhirayha Formulation and skin penetration study of lyotropicliquid crystal incorporating mulberry stem (Morus alba L)extract [MS thesis] Naresuan University Phitsanulok Thai-land 2013

[8] M Pudla K Limposuwan and P Utaisincharoen ldquoBurkholde-ria pseudomallei-Induced expression of a negative regulatorsterile-120572 and Armadillo motif-containing protein in mousemacrophages a possible mechanism for suppression of theMyD88-independent pathwayrdquo Infection and Immunity vol 79no 7 pp 2921ndash2927 2011

[9] N Soonthornsit and T Pitaksuteepong ldquoMicroemulsion for-mulation containing mulberry stem extractrdquo in Proceedings ofthe 38th Congress on Science and Technology of Thailand 6pages Chiang Mai Thailand October 2012

[10] C H Hong S K Hur O-J Oh S S Kim K A Nam and S KLee ldquoEvaluation of natural products on inhibition of induciblecyclooxygenase (COX-2) and nitric oxide synthase (iNOS) incultured mouse macrophage cellsrdquo Journal of Ethnopharmacol-ogy vol 83 no 1-2 pp 153ndash159 2002

[11] E-M Choi and J-K Hwang ldquoEffects of Morus alba leafextract on the production of nitric oxide prostaglandin E2 andcytokines in RAW2647 macrophagesrdquo Fitoterapia vol 76 no7-8 pp 608ndash613 2005

[12] Z-G Yang K Matsuzaki S Takamatsu and S KitanakaldquoInhibitory effects of constituents from Morus alba var mul-ticaulis on Differentiation of 3T3-L1 cells and nitric Oxide

8 Evidence-Based Complementary and Alternative Medicine

Production in RAW2647 Cellsrdquo Molecules vol 16 no 7 pp6010ndash6022 2011

[13] H J Lim H-G Jin E-R Woo S K Lee and H P Kim ldquoTheroot barks ofMorus alba and the flavonoid constituents inhibitairway inflammationrdquo Journal of Ethnopharmacology vol 149no 1 pp 169ndash175 2013

[14] E Park S-M Lee J E Lee and J-H Kim ldquoAnti-inflammatoryactivity of mulberry leaf extract through inhibition of NF-120581BrdquoJournal of Functional Foods vol 5 no 1 pp 178ndash186 2013

[15] H Zelova Z Hanakova Z Cermakova et al ldquoEvaluation ofanti-inflammatory activity of prenylated substances isolatedfromMorus alba andMorus nigrardquo Journal of Natural Productsvol 77 no 6 pp 1297ndash1303 2014

[16] A Abate S Oberle and H Schroder ldquoLipopolysaccharide-induced expression of cyclooxygenase-2 inmousemacrophagesis inhibited by chloromethylketones and a direct inhibitor ofNF-120581B translocationrdquo Prostaglandins and Other Lipid Media-tors vol 56 no 5-6 pp 277ndash290 1998

[17] S-H Lee C-HKwak S-K Lee et al ldquoAnti-inflammatory effectof ascochlorin in lps-stimulated raw 2647 macrophage cells isaccompanied with the down-regulation of iNOS COX-2 andproinflammatory cytokines through NF-120581B ERK12 and p38signaling pathwayrdquo Journal of Cellular Biochemistry vol 117 no4 pp 978ndash987 2016

[18] L Parente ldquoPros and Cons of Selective Inhibition of COX-2versus Dual LipoxygenaseCOX Inhibition Is Two Better thanOnerdquo The Journal of Rheumatology vol 28 no 11 pp 2375ndash2382 2001

[19] U N Das ldquoCyclooxygenase (COX) lipoxygenase (LO) path-ways and generation of lipoxins resolvins protections andmaresinsrdquo in Molecular Basis of Health and Disease N DUndurti Ed vol 4 pp 59ndash61 Springer Science BusinessMediaBV Dordrecht Holland 2010

[20] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[21] C Riviere S Krisa L Pechamat et al ldquoPolyphenols from thestems of Morus alba and their inhibitory activity against nitricoxide production by lipopolysaccharide-activated microgliardquoFitoterapia vol 97 pp 253ndash260 2014

[22] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation and Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

to hyperfilm (GE Healthcare Buckinghamshire UK) TheiNOS immunoblot signals were compared with 120573-actin andcalculated as the relative protein expression

255 NO Production Assay As detailed above the nitriteconcentration in cell supernatant was measured as an indica-tor of NO production using the Griess Reagent Kit Briefly150 120583l of each supernatant was mixed with 20120583l of Griessreagent (1 sulfanilamide in 5 phosphoric and 01 naph-thylethylenediamine dihydrochloride in water) and 130 120583l ofdistilledwater in a 96-well plateThemixtures were incubatedat room temperature for 30min and then the absorbanceof each well was determined at 548 nm using a microplatereader The amount of nitrite in samples was back-calculatedfrom a sodium nitrite calibration curve (0ndash100 120583M)

26 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) Results were obtained from at leastthree independent experiments each performed in triplicateStatistical analysis was determined using one-way analysis ofvariance (ANOVA) followed by the Tukeyrsquos test for multiplecomparisons (GraphPad Prism 60 GraphPad Software IncSan Diego USA) 119875 values less than or equal to 005 wereconsidered significant

3 Results and Discussion

The leaves fruit and bark of mulberry have long been usedto treat various illnesses and to improve health functionsHowever few reports have investigated the health benefits ofthe stem wood of mulberry which is abundantly available inThailand The anti-inflammatory effects were the biologicalactivity particularly investigated in this study

31 HPLC Analysis of Oxyresveratrol in the MSE First theMSE was prepared Ethanolic extract was obtained as apowder which had a dark brown color with a percentageyield of 513 of the raw woody material The amount ofoxyresveratrol in the MSE was analyzed by HPLC Thecalculated amount of oxyresveratrol in the MSE was 1787 plusmn061 [9]

32 TLC Analysis TLC analysis was performed to conductquality control of natural herbal product containing complexmixtures of compounds like in the case of mulberry stemextract Oxyresveratrol one of the bioactive compoundsfound in mulberry stem extract was used as a markerObserving under UV light at 254 nm and 366 nm the resultsshowed that mulberry stem extract showed eight spots with119877119891 values 002 009 016 044 064 071 078 and 087 whileoxyresveratrol showed only clear one spot with 119877119891 value 044by comparing 119877119891 value It was clearly shown that a reddishspot observed in mulberry stem extract after spraying TLCplate with anisaldehyde reagent with 119877119891 value of 044 wasoxyresveratrol (Figure 1)

33 Cell Viability To ensure that the cells were healthy beforeperforming the bioactivity assays and the tested concentra-tions were not toxic to the cells cell viability after treatment

Spraying with anisaldehyde reagent

Mulberry stem extractOxyresveratrol

UV light (휆 = 254 nm)UV light (휆 = 366 nm)

Figure 1 TLC fingerprint of mulberry stem extract Standardoxyresveratrol was used as a marker Mobile phase system wasmethanol chloroform (15 85 vv)

with various concentrations of the MSE was determinedThe concentrations of the MSE that yielded cell viabilityhigher than 80 were the main aim of this experiment TheRAW 2647 cell viability slightly decreased with increasingthe concentration of theMSE from 10 to 150120583gml (Figure 2)Identifying the concentrations of the MSE that yielded cellviability higher than 80 it was found to be in the range of10 to 40 120583gml and the concentrations of the MSE selectedfor subsequent studies were 20 and 40120583gml Oxyresveratrolwas included in this study as the marker of the MSE Anequivalent amount of oxyresveratrol in each concentrationof the extract tested (for example equivalent amount ofoxyresveratrol in the extract 100120583gml was 17 120583gml) wasalso evaluated for the effect on cell viability The resultswere shown to be similar to those observed in the MSE-treated cells (Figure 2) The anti-inflammatory effects ofoxyresveratrol were also confirmed in subsequent studies inwhich the selected concentrations of oxyresveratrol were 5and 10 120583gml

34 Anti-Inflammatory Activity of the MSE M alba extracthas been demonstrated to exhibit anti-inflammatory activitythrough the inhibition of iNOS NO and COX [10ndash15]However most of the extract tested was prepared from leaves[10 11 14] and root bark [12 13 15] In our study anti-inflammation activity of the extract prepared from mulberrystems was investigated

In an LPS-stimulated mouse macrophage RAW 2647cells model the NF-120581B signaling pathway plays a crucialrole in regulating inflammation through the transcriptionof iNOS COX and cytokine genes Found in the cyto-plasm of resting cells dimer NF-120581B is normally confinedto an inactive cytoplasmic complex through binding to aninhibitory protein I-120581B which masks its nuclear localizationsignal Exposure of cells to external proinflammatory stimulisuch as mitogens inflammatory cytokines and LPS causesrapid I-120581B phosphorylation by I-120581B kinase (IKK) resultingin free and subsequent nuclear translocation of NF-120581B [16

Evidence-Based Complementary and Alternative Medicine 5

010

10

20

20

30

30

40

40

50

50

60708090

100

100 150

MSE Oxyresveratrol

ce

ll vi

abili

ty

17 17minus + + + + + + + + + + + + + + +

34 51 68 85 255

l(휇gml)LPS 100 ngml

Figure 2 The percentage viability of RAW 2647 cells which was untreated (negative control) treated with only 100 ngml LPS (positivecontrol) and treated with various concentrations of MSE and oxyresveratrol in the presence of 100 ngml LPS for 24 h The viability of cellswas determined by MTT assay The data were represented as means plusmn SD of three independent experiments

iNOS

minus +++++

(휇gml)20 40 105

MSE Oxyresveratrol

β-Actin

LPS 100 ngml

(a)

lowastlowast

Rela

tive e

xpre

ssio

nle

vels

of iN

OS

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

080

060

040

020

000LPS 100 ngml

(b)

Figure 3 The effects of MSE and oxyresveratrol on the expression of iNOS mRNA in RAW 2647 cells The untreated cells were used asa negative control and the cells treated with only 100 ngml LPS were served as a positive control (a) RT-PCR analysis for detection ofiNOS mRNA expression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml oroxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS mRNA compared to 120573-actin The data wererepresented as means plusmn SD of three independent experiments The means marked with lowast are significantly different (119875 lt 005) from that ofthe cells treated with only LPS

17] In the nucleus NF-120581B induces the transcription of alarge variety of target proinflammatory genes including iNOSand COX-2 iNOS then further facilitates the conversionof L-arginine to L-citrulline and a large amount of NOwhile COX-2 catalyzes the conversion of arachidonic acid toprostaglandins Prostaglandins (PGs) especially PGE2 andPGI2 cause vasodilation that contribute to the vascular signsof inflammation and potentiate edema formation [18 19]

341 Effect of the MSE on the Expression of iNOS mRNAand Protein The anti-inflammatory effects of the MSE at theconcentrations of 20 and 40120583gml were investigated throughthe inhibition on the expression of iNOS mRNA and itsprotein as well as the production of NO Oxyresveratrol(marker) at the concentrations of 5 and 10 120583gml was alsotested 120573-Actin was used as an internal standard for RT-PCRassay As expected the expression of iNOS mRNA and itsproteinwas detected by stimulatingRAW2647 cells with LPS(Figures 3 and 4)The expression level of iNOS in cells treatedwith theMSE at the concentration of 40 120583gml was decreased

by 50 compared with that in cells treated with LPS alone(Figure 3) Similar results were observed in cells treated withoxyresveratrol

Western blotting was further performed to confirm theeffects of the MSE on iNOS protein expression The resultsshowed that iNOS protein was significantly decreased inuntreated RAW 2647 cells comparing with LPS-treatedRAW 2647 cells (Figure 4) Therefore the validity of theiNOS protein assay was again confirmed The MSE at theconcentrations of 20 (P lt 005) and 40 (P lt 0001)120583gml andoxyresveratrol at 10 (P lt 005) 120583gml significantly inhibitedthe expression of iNOS protein compared with the cellstreated only with LPS

342 Effect of the MSE on NO Production The inducibleforms of NOS are the most important proinflammatoryenzymes responsible for increasing the levels of NO There-fore the effect of tested compounds on the inhibition ofNO production was further investigated However NO inthe biological matrix is very unstable and rapidly oxidizes

6 Evidence-Based Complementary and Alternative Medicine

MSE Oxyresveratrol

minus +++++

20 40 10 (휇gml)5

iNOS

β-Actin

LPS 100 ngml

(a)

000

020

040

060

080

100

MSE

lowast lowast

lowastlowastlowast

Rela

tive e

xpre

ssio

n le

vels

of iN

OS

prot

ein

minus +++++

20 40 10 (휇gml)5

Oxyresveratrol

LPS 100 ngml

(b)

Figure 4 The effects of MSE and oxyresveratrol on the expression of iNOS protein in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) Immunoblotting analysis for determinationthe expression of iNOS protein in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS protein compared to 120573-actin The datawere represented as means plusmn SD of three independent experiments The means marked with lowast lowastlowastlowast are significantly different (119875 lt 005) and(119875 lt 0001) respectively from that of the cells treated with only LPS

Nitr

ite (휇

M)

minus +++++

20 40 10 (휇gml)5

MSE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Oxyresveratrol

2500

2000

1500

1000

500

000

LPS 100 ngml

Figure 5 The effects of MSE and oxyresveratrol on the productionof nitrite in LPS-stimulated RAW 2647 cells which were untreated(negative control) treated with only 100 ngml LPS (positive con-trol) pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations of 5 and 10120583gml for 2 hand then treated with 100 ngml LPS for 16 h The supernatant wascollected and determined by Griess reaction assay The data wererepresented as means plusmn SD of three independent experiments Themeansmarkedwithlowastlowastlowastlowastlowastlowast are significantly different (119875 lt 005)(119875 lt 0005) and (119875 lt 0001) respectively from that of the cellstreated with only LPS

to nitrite (NO2minus) and thus the measurement of nitrite is

routinely used as an index of NO productionFor the untreated RAW 2647 cells the concentration

of nitrite could not be detected (Figure 5) Once the cellswere stimulated with LPS a high concentration of nitrite wasproduced The MSE at concentrations of 20 and 40 120583gmlwere shown to inhibit nitrite production by 34 and 54respectively and oxyresveratrol at concentrations of 5 and10 120583gml was able to inhibit nitrite production by 16 and28 respectively (Figure 5)

343 Effect of the MSE on COX-2 mRNA Expression Asshown in Figure 6 theMSE was able to inhibit the expression

of COX-2 mRNA in LPS-activated RAW 2647 cells Theinhibitory activity of the MSE at 40 120583gml was shown to behigher than that of the MSE at 20 120583gmlThe expression levelof COX-2 mRNA in cells treated with the MSE at 40 120583gmldecreased by 70 compared with that in cells treated withLPS alone Similar results were observed when LPS-activatedRAW 2647 cells were incubated with oxyresveratrol

The results observed in this study clearly showed that theMSE could inhibit NO production through the inhibitionof both iNOS mRNA and protein expression In additionthe extract was shown to suppress the expression of COX-2 mRNA in LPS-stimulated RAW 2647 cells The inhibitioneffect on production of iNOS COX-2 and NO of the MSEat 40 120583gml was found to be higher than that of the MSE at20120583gml The MSE showed more potent inhibitory effectsthan oxyresveratrol at an equivalent amount of oxyresver-atrol (the MSE at 40 120583gml equivalent to 68120583gml) It hasbeen recently reported that the stems of M alba containedflavonoids (morusin) stilbenoids (mulberroside resveratroland oxyresveratrol) and coumarins [1] These polyphenolswere shown to possess anti-inflammatory effects [20ndash22]Wealso determined the amounts of total phenolic of the extractand it was 3487 plusmn 112120583mol GAEmg extract Thereforethese polyphenols other than oxyresveratrol may explainthe superior anti-inflammatory effect of the MSE Althoughthe precise molecular mechanisms responsible for the anti-inflammatory activities of theMSE cannot be explained in thepresent study oxyresveratrol which is an active componentpresented in the extract has been reported to exhibit its anti-inflammatory effect via the inhibition of the NF-120581B and theMAPK pathways [2 3] However further elucidation of theinsight mechanisms involved the anti-inflammatory effect ofthis plant extract is required

4 Conclusions

This study is the first to demonstrate the potential anti-inflammatory effects of the extract prepared from mulberry

Evidence-Based Complementary and Alternative Medicine 7

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

COX-2

β-Actin

LPS 100 ngml

(a)

000

030

060

090

120

150

Relat

ive e

xpre

ssio

n le

vels

of COX-2

minus +++++

20 40 10 (휇gml)5

lowastlowastlowast

lowastlowast lowastlowast

MSE Oxyresveratrol

LPS 100 ngml

(b)

Figure 6 The effects of MSE and oxyresveratrol on the expression of COX-2 mRNA in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) RT-PCR analysis for detection of COX-2mRNAexpression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml or oxyresveratrolat the concentrations of 5 and 10120583gml for 6 h (b) The relative expression of COX-2 mRNA compared to 120573-actin The data were representedas means plusmn SD of three independent experiments The means marked with lowastlowast lowastlowastlowast are significantly different (119875 lt 0005) and (119875 lt 0001)respectively from that of the cells treated with only LPS

stems on LPS-induced RAW 2647 cells The investigationfound that concentrations at 20 and 40 120583gml had no toxiceffect The results suggest that the stem extract mediates itsanti-inflammatory activities through the inhibition of iNOSNO and COX-2 production Further studies are necessary tofully define the precise mechanisms involved

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors would like to acknowledge the financial supportfrom the yearly budget of Naresuan University Thailand(R2555B069 and R2558B053) the Center for Innovation inChemistry (PERCH-CIC) and National Research Council ofThailand Graduate Student Scholarship (2014) They expressspecial gratitude towards theQueen Sirikit SericultureCenter(Tak) for the supply of plant materials and to Mr CheinYhirayha for the purification of the oxyresveratrol Manythanks are due toMr RoyMorien of the Naresuan UniversityLanguage Centre for his editing assistance and advice onEnglish expression in this manuscript

References

[1] E W-C Chan P-Y Lye and S-K Wong ldquoPhytochemistrypharmacology and clinical trials of Morus albardquo Chinese Jour-nal of Natural Medicines vol 14 no 1 pp 17ndash30 2016

[2] K-O Chung B-Y Kim M-H Lee et al ldquoIn-vitro and in-vivoanti-inflammatory effect of oxyresveratrol fromMorus alba LrdquoJournal of Pharmacy and Pharmacology vol 55 no 12 pp 1695ndash1700 2003

[3] H S Lee D H Kim J E Hong J-Y Lee and E JKim ldquoOxyresveratrol suppresses lipopolysaccharide-induced

inflammatory responses in murine macrophagesrdquo Human andExperimental Toxicology vol 34 no 8 pp 808ndash818 2015

[4] P Thongsuk In vitro and clinical study of mulberry extractfor skin whitening product [MS thesis] Naresuan UniversityPhitsanulok Thailand 2007

[5] D Laveti M Kumar and R Hemalatha ldquoAnti-inflammatorytreatments for chronic diseases a reviewrdquo Inflammation ampAllergymdashDrug Targets vol 12 no 5 pp 349ndash361 2013

[6] Y-C Chen Y-J Tien C-H Chen et al ldquoMorus alba and activecompound oxyresveratrol exert anti-inflammatory activity viainhibition of leukocyte migration involving MEKERK signal-ingrdquo BMCComplementary and AlternativeMedicine vol 13 no1 pp 45ndash55 2013

[7] C Yhirayha Formulation and skin penetration study of lyotropicliquid crystal incorporating mulberry stem (Morus alba L)extract [MS thesis] Naresuan University Phitsanulok Thai-land 2013

[8] M Pudla K Limposuwan and P Utaisincharoen ldquoBurkholde-ria pseudomallei-Induced expression of a negative regulatorsterile-120572 and Armadillo motif-containing protein in mousemacrophages a possible mechanism for suppression of theMyD88-independent pathwayrdquo Infection and Immunity vol 79no 7 pp 2921ndash2927 2011

[9] N Soonthornsit and T Pitaksuteepong ldquoMicroemulsion for-mulation containing mulberry stem extractrdquo in Proceedings ofthe 38th Congress on Science and Technology of Thailand 6pages Chiang Mai Thailand October 2012

[10] C H Hong S K Hur O-J Oh S S Kim K A Nam and S KLee ldquoEvaluation of natural products on inhibition of induciblecyclooxygenase (COX-2) and nitric oxide synthase (iNOS) incultured mouse macrophage cellsrdquo Journal of Ethnopharmacol-ogy vol 83 no 1-2 pp 153ndash159 2002

[11] E-M Choi and J-K Hwang ldquoEffects of Morus alba leafextract on the production of nitric oxide prostaglandin E2 andcytokines in RAW2647 macrophagesrdquo Fitoterapia vol 76 no7-8 pp 608ndash613 2005

[12] Z-G Yang K Matsuzaki S Takamatsu and S KitanakaldquoInhibitory effects of constituents from Morus alba var mul-ticaulis on Differentiation of 3T3-L1 cells and nitric Oxide

8 Evidence-Based Complementary and Alternative Medicine

Production in RAW2647 Cellsrdquo Molecules vol 16 no 7 pp6010ndash6022 2011

[13] H J Lim H-G Jin E-R Woo S K Lee and H P Kim ldquoTheroot barks ofMorus alba and the flavonoid constituents inhibitairway inflammationrdquo Journal of Ethnopharmacology vol 149no 1 pp 169ndash175 2013

[14] E Park S-M Lee J E Lee and J-H Kim ldquoAnti-inflammatoryactivity of mulberry leaf extract through inhibition of NF-120581BrdquoJournal of Functional Foods vol 5 no 1 pp 178ndash186 2013

[15] H Zelova Z Hanakova Z Cermakova et al ldquoEvaluation ofanti-inflammatory activity of prenylated substances isolatedfromMorus alba andMorus nigrardquo Journal of Natural Productsvol 77 no 6 pp 1297ndash1303 2014

[16] A Abate S Oberle and H Schroder ldquoLipopolysaccharide-induced expression of cyclooxygenase-2 inmousemacrophagesis inhibited by chloromethylketones and a direct inhibitor ofNF-120581B translocationrdquo Prostaglandins and Other Lipid Media-tors vol 56 no 5-6 pp 277ndash290 1998

[17] S-H Lee C-HKwak S-K Lee et al ldquoAnti-inflammatory effectof ascochlorin in lps-stimulated raw 2647 macrophage cells isaccompanied with the down-regulation of iNOS COX-2 andproinflammatory cytokines through NF-120581B ERK12 and p38signaling pathwayrdquo Journal of Cellular Biochemistry vol 117 no4 pp 978ndash987 2016

[18] L Parente ldquoPros and Cons of Selective Inhibition of COX-2versus Dual LipoxygenaseCOX Inhibition Is Two Better thanOnerdquo The Journal of Rheumatology vol 28 no 11 pp 2375ndash2382 2001

[19] U N Das ldquoCyclooxygenase (COX) lipoxygenase (LO) path-ways and generation of lipoxins resolvins protections andmaresinsrdquo in Molecular Basis of Health and Disease N DUndurti Ed vol 4 pp 59ndash61 Springer Science BusinessMediaBV Dordrecht Holland 2010

[20] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[21] C Riviere S Krisa L Pechamat et al ldquoPolyphenols from thestems of Morus alba and their inhibitory activity against nitricoxide production by lipopolysaccharide-activated microgliardquoFitoterapia vol 97 pp 253ndash260 2014

[22] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation and Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

010

10

20

20

30

30

40

40

50

50

60708090

100

100 150

MSE Oxyresveratrol

ce

ll vi

abili

ty

17 17minus + + + + + + + + + + + + + + +

34 51 68 85 255

l(휇gml)LPS 100 ngml

Figure 2 The percentage viability of RAW 2647 cells which was untreated (negative control) treated with only 100 ngml LPS (positivecontrol) and treated with various concentrations of MSE and oxyresveratrol in the presence of 100 ngml LPS for 24 h The viability of cellswas determined by MTT assay The data were represented as means plusmn SD of three independent experiments

iNOS

minus +++++

(휇gml)20 40 105

MSE Oxyresveratrol

β-Actin

LPS 100 ngml

(a)

lowastlowast

Rela

tive e

xpre

ssio

nle

vels

of iN

OS

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

080

060

040

020

000LPS 100 ngml

(b)

Figure 3 The effects of MSE and oxyresveratrol on the expression of iNOS mRNA in RAW 2647 cells The untreated cells were used asa negative control and the cells treated with only 100 ngml LPS were served as a positive control (a) RT-PCR analysis for detection ofiNOS mRNA expression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml oroxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS mRNA compared to 120573-actin The data wererepresented as means plusmn SD of three independent experiments The means marked with lowast are significantly different (119875 lt 005) from that ofthe cells treated with only LPS

17] In the nucleus NF-120581B induces the transcription of alarge variety of target proinflammatory genes including iNOSand COX-2 iNOS then further facilitates the conversionof L-arginine to L-citrulline and a large amount of NOwhile COX-2 catalyzes the conversion of arachidonic acid toprostaglandins Prostaglandins (PGs) especially PGE2 andPGI2 cause vasodilation that contribute to the vascular signsof inflammation and potentiate edema formation [18 19]

341 Effect of the MSE on the Expression of iNOS mRNAand Protein The anti-inflammatory effects of the MSE at theconcentrations of 20 and 40120583gml were investigated throughthe inhibition on the expression of iNOS mRNA and itsprotein as well as the production of NO Oxyresveratrol(marker) at the concentrations of 5 and 10 120583gml was alsotested 120573-Actin was used as an internal standard for RT-PCRassay As expected the expression of iNOS mRNA and itsproteinwas detected by stimulatingRAW2647 cells with LPS(Figures 3 and 4)The expression level of iNOS in cells treatedwith theMSE at the concentration of 40 120583gml was decreased

by 50 compared with that in cells treated with LPS alone(Figure 3) Similar results were observed in cells treated withoxyresveratrol

Western blotting was further performed to confirm theeffects of the MSE on iNOS protein expression The resultsshowed that iNOS protein was significantly decreased inuntreated RAW 2647 cells comparing with LPS-treatedRAW 2647 cells (Figure 4) Therefore the validity of theiNOS protein assay was again confirmed The MSE at theconcentrations of 20 (P lt 005) and 40 (P lt 0001)120583gml andoxyresveratrol at 10 (P lt 005) 120583gml significantly inhibitedthe expression of iNOS protein compared with the cellstreated only with LPS

342 Effect of the MSE on NO Production The inducibleforms of NOS are the most important proinflammatoryenzymes responsible for increasing the levels of NO There-fore the effect of tested compounds on the inhibition ofNO production was further investigated However NO inthe biological matrix is very unstable and rapidly oxidizes

6 Evidence-Based Complementary and Alternative Medicine

MSE Oxyresveratrol

minus +++++

20 40 10 (휇gml)5

iNOS

β-Actin

LPS 100 ngml

(a)

000

020

040

060

080

100

MSE

lowast lowast

lowastlowastlowast

Rela

tive e

xpre

ssio

n le

vels

of iN

OS

prot

ein

minus +++++

20 40 10 (휇gml)5

Oxyresveratrol

LPS 100 ngml

(b)

Figure 4 The effects of MSE and oxyresveratrol on the expression of iNOS protein in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) Immunoblotting analysis for determinationthe expression of iNOS protein in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS protein compared to 120573-actin The datawere represented as means plusmn SD of three independent experiments The means marked with lowast lowastlowastlowast are significantly different (119875 lt 005) and(119875 lt 0001) respectively from that of the cells treated with only LPS

Nitr

ite (휇

M)

minus +++++

20 40 10 (휇gml)5

MSE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Oxyresveratrol

2500

2000

1500

1000

500

000

LPS 100 ngml

Figure 5 The effects of MSE and oxyresveratrol on the productionof nitrite in LPS-stimulated RAW 2647 cells which were untreated(negative control) treated with only 100 ngml LPS (positive con-trol) pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations of 5 and 10120583gml for 2 hand then treated with 100 ngml LPS for 16 h The supernatant wascollected and determined by Griess reaction assay The data wererepresented as means plusmn SD of three independent experiments Themeansmarkedwithlowastlowastlowastlowastlowastlowast are significantly different (119875 lt 005)(119875 lt 0005) and (119875 lt 0001) respectively from that of the cellstreated with only LPS

to nitrite (NO2minus) and thus the measurement of nitrite is

routinely used as an index of NO productionFor the untreated RAW 2647 cells the concentration

of nitrite could not be detected (Figure 5) Once the cellswere stimulated with LPS a high concentration of nitrite wasproduced The MSE at concentrations of 20 and 40 120583gmlwere shown to inhibit nitrite production by 34 and 54respectively and oxyresveratrol at concentrations of 5 and10 120583gml was able to inhibit nitrite production by 16 and28 respectively (Figure 5)

343 Effect of the MSE on COX-2 mRNA Expression Asshown in Figure 6 theMSE was able to inhibit the expression

of COX-2 mRNA in LPS-activated RAW 2647 cells Theinhibitory activity of the MSE at 40 120583gml was shown to behigher than that of the MSE at 20 120583gmlThe expression levelof COX-2 mRNA in cells treated with the MSE at 40 120583gmldecreased by 70 compared with that in cells treated withLPS alone Similar results were observed when LPS-activatedRAW 2647 cells were incubated with oxyresveratrol

The results observed in this study clearly showed that theMSE could inhibit NO production through the inhibitionof both iNOS mRNA and protein expression In additionthe extract was shown to suppress the expression of COX-2 mRNA in LPS-stimulated RAW 2647 cells The inhibitioneffect on production of iNOS COX-2 and NO of the MSEat 40 120583gml was found to be higher than that of the MSE at20120583gml The MSE showed more potent inhibitory effectsthan oxyresveratrol at an equivalent amount of oxyresver-atrol (the MSE at 40 120583gml equivalent to 68120583gml) It hasbeen recently reported that the stems of M alba containedflavonoids (morusin) stilbenoids (mulberroside resveratroland oxyresveratrol) and coumarins [1] These polyphenolswere shown to possess anti-inflammatory effects [20ndash22]Wealso determined the amounts of total phenolic of the extractand it was 3487 plusmn 112120583mol GAEmg extract Thereforethese polyphenols other than oxyresveratrol may explainthe superior anti-inflammatory effect of the MSE Althoughthe precise molecular mechanisms responsible for the anti-inflammatory activities of theMSE cannot be explained in thepresent study oxyresveratrol which is an active componentpresented in the extract has been reported to exhibit its anti-inflammatory effect via the inhibition of the NF-120581B and theMAPK pathways [2 3] However further elucidation of theinsight mechanisms involved the anti-inflammatory effect ofthis plant extract is required

4 Conclusions

This study is the first to demonstrate the potential anti-inflammatory effects of the extract prepared from mulberry

Evidence-Based Complementary and Alternative Medicine 7

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

COX-2

β-Actin

LPS 100 ngml

(a)

000

030

060

090

120

150

Relat

ive e

xpre

ssio

n le

vels

of COX-2

minus +++++

20 40 10 (휇gml)5

lowastlowastlowast

lowastlowast lowastlowast

MSE Oxyresveratrol

LPS 100 ngml

(b)

Figure 6 The effects of MSE and oxyresveratrol on the expression of COX-2 mRNA in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) RT-PCR analysis for detection of COX-2mRNAexpression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml or oxyresveratrolat the concentrations of 5 and 10120583gml for 6 h (b) The relative expression of COX-2 mRNA compared to 120573-actin The data were representedas means plusmn SD of three independent experiments The means marked with lowastlowast lowastlowastlowast are significantly different (119875 lt 0005) and (119875 lt 0001)respectively from that of the cells treated with only LPS

stems on LPS-induced RAW 2647 cells The investigationfound that concentrations at 20 and 40 120583gml had no toxiceffect The results suggest that the stem extract mediates itsanti-inflammatory activities through the inhibition of iNOSNO and COX-2 production Further studies are necessary tofully define the precise mechanisms involved

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors would like to acknowledge the financial supportfrom the yearly budget of Naresuan University Thailand(R2555B069 and R2558B053) the Center for Innovation inChemistry (PERCH-CIC) and National Research Council ofThailand Graduate Student Scholarship (2014) They expressspecial gratitude towards theQueen Sirikit SericultureCenter(Tak) for the supply of plant materials and to Mr CheinYhirayha for the purification of the oxyresveratrol Manythanks are due toMr RoyMorien of the Naresuan UniversityLanguage Centre for his editing assistance and advice onEnglish expression in this manuscript

References

[1] E W-C Chan P-Y Lye and S-K Wong ldquoPhytochemistrypharmacology and clinical trials of Morus albardquo Chinese Jour-nal of Natural Medicines vol 14 no 1 pp 17ndash30 2016

[2] K-O Chung B-Y Kim M-H Lee et al ldquoIn-vitro and in-vivoanti-inflammatory effect of oxyresveratrol fromMorus alba LrdquoJournal of Pharmacy and Pharmacology vol 55 no 12 pp 1695ndash1700 2003

[3] H S Lee D H Kim J E Hong J-Y Lee and E JKim ldquoOxyresveratrol suppresses lipopolysaccharide-induced

inflammatory responses in murine macrophagesrdquo Human andExperimental Toxicology vol 34 no 8 pp 808ndash818 2015

[4] P Thongsuk In vitro and clinical study of mulberry extractfor skin whitening product [MS thesis] Naresuan UniversityPhitsanulok Thailand 2007

[5] D Laveti M Kumar and R Hemalatha ldquoAnti-inflammatorytreatments for chronic diseases a reviewrdquo Inflammation ampAllergymdashDrug Targets vol 12 no 5 pp 349ndash361 2013

[6] Y-C Chen Y-J Tien C-H Chen et al ldquoMorus alba and activecompound oxyresveratrol exert anti-inflammatory activity viainhibition of leukocyte migration involving MEKERK signal-ingrdquo BMCComplementary and AlternativeMedicine vol 13 no1 pp 45ndash55 2013

[7] C Yhirayha Formulation and skin penetration study of lyotropicliquid crystal incorporating mulberry stem (Morus alba L)extract [MS thesis] Naresuan University Phitsanulok Thai-land 2013

[8] M Pudla K Limposuwan and P Utaisincharoen ldquoBurkholde-ria pseudomallei-Induced expression of a negative regulatorsterile-120572 and Armadillo motif-containing protein in mousemacrophages a possible mechanism for suppression of theMyD88-independent pathwayrdquo Infection and Immunity vol 79no 7 pp 2921ndash2927 2011

[9] N Soonthornsit and T Pitaksuteepong ldquoMicroemulsion for-mulation containing mulberry stem extractrdquo in Proceedings ofthe 38th Congress on Science and Technology of Thailand 6pages Chiang Mai Thailand October 2012

[10] C H Hong S K Hur O-J Oh S S Kim K A Nam and S KLee ldquoEvaluation of natural products on inhibition of induciblecyclooxygenase (COX-2) and nitric oxide synthase (iNOS) incultured mouse macrophage cellsrdquo Journal of Ethnopharmacol-ogy vol 83 no 1-2 pp 153ndash159 2002

[11] E-M Choi and J-K Hwang ldquoEffects of Morus alba leafextract on the production of nitric oxide prostaglandin E2 andcytokines in RAW2647 macrophagesrdquo Fitoterapia vol 76 no7-8 pp 608ndash613 2005

[12] Z-G Yang K Matsuzaki S Takamatsu and S KitanakaldquoInhibitory effects of constituents from Morus alba var mul-ticaulis on Differentiation of 3T3-L1 cells and nitric Oxide

8 Evidence-Based Complementary and Alternative Medicine

Production in RAW2647 Cellsrdquo Molecules vol 16 no 7 pp6010ndash6022 2011

[13] H J Lim H-G Jin E-R Woo S K Lee and H P Kim ldquoTheroot barks ofMorus alba and the flavonoid constituents inhibitairway inflammationrdquo Journal of Ethnopharmacology vol 149no 1 pp 169ndash175 2013

[14] E Park S-M Lee J E Lee and J-H Kim ldquoAnti-inflammatoryactivity of mulberry leaf extract through inhibition of NF-120581BrdquoJournal of Functional Foods vol 5 no 1 pp 178ndash186 2013

[15] H Zelova Z Hanakova Z Cermakova et al ldquoEvaluation ofanti-inflammatory activity of prenylated substances isolatedfromMorus alba andMorus nigrardquo Journal of Natural Productsvol 77 no 6 pp 1297ndash1303 2014

[16] A Abate S Oberle and H Schroder ldquoLipopolysaccharide-induced expression of cyclooxygenase-2 inmousemacrophagesis inhibited by chloromethylketones and a direct inhibitor ofNF-120581B translocationrdquo Prostaglandins and Other Lipid Media-tors vol 56 no 5-6 pp 277ndash290 1998

[17] S-H Lee C-HKwak S-K Lee et al ldquoAnti-inflammatory effectof ascochlorin in lps-stimulated raw 2647 macrophage cells isaccompanied with the down-regulation of iNOS COX-2 andproinflammatory cytokines through NF-120581B ERK12 and p38signaling pathwayrdquo Journal of Cellular Biochemistry vol 117 no4 pp 978ndash987 2016

[18] L Parente ldquoPros and Cons of Selective Inhibition of COX-2versus Dual LipoxygenaseCOX Inhibition Is Two Better thanOnerdquo The Journal of Rheumatology vol 28 no 11 pp 2375ndash2382 2001

[19] U N Das ldquoCyclooxygenase (COX) lipoxygenase (LO) path-ways and generation of lipoxins resolvins protections andmaresinsrdquo in Molecular Basis of Health and Disease N DUndurti Ed vol 4 pp 59ndash61 Springer Science BusinessMediaBV Dordrecht Holland 2010

[20] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[21] C Riviere S Krisa L Pechamat et al ldquoPolyphenols from thestems of Morus alba and their inhibitory activity against nitricoxide production by lipopolysaccharide-activated microgliardquoFitoterapia vol 97 pp 253ndash260 2014

[22] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation and Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

MSE Oxyresveratrol

minus +++++

20 40 10 (휇gml)5

iNOS

β-Actin

LPS 100 ngml

(a)

000

020

040

060

080

100

MSE

lowast lowast

lowastlowastlowast

Rela

tive e

xpre

ssio

n le

vels

of iN

OS

prot

ein

minus +++++

20 40 10 (휇gml)5

Oxyresveratrol

LPS 100 ngml

(b)

Figure 4 The effects of MSE and oxyresveratrol on the expression of iNOS protein in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) Immunoblotting analysis for determinationthe expression of iNOS protein in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations 5 and 10 120583gml for 2 h (b) The relative expression of iNOS protein compared to 120573-actin The datawere represented as means plusmn SD of three independent experiments The means marked with lowast lowastlowastlowast are significantly different (119875 lt 005) and(119875 lt 0001) respectively from that of the cells treated with only LPS

Nitr

ite (휇

M)

minus +++++

20 40 10 (휇gml)5

MSE

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Oxyresveratrol

2500

2000

1500

1000

500

000

LPS 100 ngml

Figure 5 The effects of MSE and oxyresveratrol on the productionof nitrite in LPS-stimulated RAW 2647 cells which were untreated(negative control) treated with only 100 ngml LPS (positive con-trol) pretreated with MSE at the concentrations of 20 and 40120583gmlor oxyresveratrol at the concentrations of 5 and 10120583gml for 2 hand then treated with 100 ngml LPS for 16 h The supernatant wascollected and determined by Griess reaction assay The data wererepresented as means plusmn SD of three independent experiments Themeansmarkedwithlowastlowastlowastlowastlowastlowast are significantly different (119875 lt 005)(119875 lt 0005) and (119875 lt 0001) respectively from that of the cellstreated with only LPS

to nitrite (NO2minus) and thus the measurement of nitrite is

routinely used as an index of NO productionFor the untreated RAW 2647 cells the concentration

of nitrite could not be detected (Figure 5) Once the cellswere stimulated with LPS a high concentration of nitrite wasproduced The MSE at concentrations of 20 and 40 120583gmlwere shown to inhibit nitrite production by 34 and 54respectively and oxyresveratrol at concentrations of 5 and10 120583gml was able to inhibit nitrite production by 16 and28 respectively (Figure 5)

343 Effect of the MSE on COX-2 mRNA Expression Asshown in Figure 6 theMSE was able to inhibit the expression

of COX-2 mRNA in LPS-activated RAW 2647 cells Theinhibitory activity of the MSE at 40 120583gml was shown to behigher than that of the MSE at 20 120583gmlThe expression levelof COX-2 mRNA in cells treated with the MSE at 40 120583gmldecreased by 70 compared with that in cells treated withLPS alone Similar results were observed when LPS-activatedRAW 2647 cells were incubated with oxyresveratrol

The results observed in this study clearly showed that theMSE could inhibit NO production through the inhibitionof both iNOS mRNA and protein expression In additionthe extract was shown to suppress the expression of COX-2 mRNA in LPS-stimulated RAW 2647 cells The inhibitioneffect on production of iNOS COX-2 and NO of the MSEat 40 120583gml was found to be higher than that of the MSE at20120583gml The MSE showed more potent inhibitory effectsthan oxyresveratrol at an equivalent amount of oxyresver-atrol (the MSE at 40 120583gml equivalent to 68120583gml) It hasbeen recently reported that the stems of M alba containedflavonoids (morusin) stilbenoids (mulberroside resveratroland oxyresveratrol) and coumarins [1] These polyphenolswere shown to possess anti-inflammatory effects [20ndash22]Wealso determined the amounts of total phenolic of the extractand it was 3487 plusmn 112120583mol GAEmg extract Thereforethese polyphenols other than oxyresveratrol may explainthe superior anti-inflammatory effect of the MSE Althoughthe precise molecular mechanisms responsible for the anti-inflammatory activities of theMSE cannot be explained in thepresent study oxyresveratrol which is an active componentpresented in the extract has been reported to exhibit its anti-inflammatory effect via the inhibition of the NF-120581B and theMAPK pathways [2 3] However further elucidation of theinsight mechanisms involved the anti-inflammatory effect ofthis plant extract is required

4 Conclusions

This study is the first to demonstrate the potential anti-inflammatory effects of the extract prepared from mulberry

Evidence-Based Complementary and Alternative Medicine 7

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

COX-2

β-Actin

LPS 100 ngml

(a)

000

030

060

090

120

150

Relat

ive e

xpre

ssio

n le

vels

of COX-2

minus +++++

20 40 10 (휇gml)5

lowastlowastlowast

lowastlowast lowastlowast

MSE Oxyresveratrol

LPS 100 ngml

(b)

Figure 6 The effects of MSE and oxyresveratrol on the expression of COX-2 mRNA in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) RT-PCR analysis for detection of COX-2mRNAexpression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml or oxyresveratrolat the concentrations of 5 and 10120583gml for 6 h (b) The relative expression of COX-2 mRNA compared to 120573-actin The data were representedas means plusmn SD of three independent experiments The means marked with lowastlowast lowastlowastlowast are significantly different (119875 lt 0005) and (119875 lt 0001)respectively from that of the cells treated with only LPS

stems on LPS-induced RAW 2647 cells The investigationfound that concentrations at 20 and 40 120583gml had no toxiceffect The results suggest that the stem extract mediates itsanti-inflammatory activities through the inhibition of iNOSNO and COX-2 production Further studies are necessary tofully define the precise mechanisms involved

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors would like to acknowledge the financial supportfrom the yearly budget of Naresuan University Thailand(R2555B069 and R2558B053) the Center for Innovation inChemistry (PERCH-CIC) and National Research Council ofThailand Graduate Student Scholarship (2014) They expressspecial gratitude towards theQueen Sirikit SericultureCenter(Tak) for the supply of plant materials and to Mr CheinYhirayha for the purification of the oxyresveratrol Manythanks are due toMr RoyMorien of the Naresuan UniversityLanguage Centre for his editing assistance and advice onEnglish expression in this manuscript

References

[1] E W-C Chan P-Y Lye and S-K Wong ldquoPhytochemistrypharmacology and clinical trials of Morus albardquo Chinese Jour-nal of Natural Medicines vol 14 no 1 pp 17ndash30 2016

[2] K-O Chung B-Y Kim M-H Lee et al ldquoIn-vitro and in-vivoanti-inflammatory effect of oxyresveratrol fromMorus alba LrdquoJournal of Pharmacy and Pharmacology vol 55 no 12 pp 1695ndash1700 2003

[3] H S Lee D H Kim J E Hong J-Y Lee and E JKim ldquoOxyresveratrol suppresses lipopolysaccharide-induced

inflammatory responses in murine macrophagesrdquo Human andExperimental Toxicology vol 34 no 8 pp 808ndash818 2015

[4] P Thongsuk In vitro and clinical study of mulberry extractfor skin whitening product [MS thesis] Naresuan UniversityPhitsanulok Thailand 2007

[5] D Laveti M Kumar and R Hemalatha ldquoAnti-inflammatorytreatments for chronic diseases a reviewrdquo Inflammation ampAllergymdashDrug Targets vol 12 no 5 pp 349ndash361 2013

[6] Y-C Chen Y-J Tien C-H Chen et al ldquoMorus alba and activecompound oxyresveratrol exert anti-inflammatory activity viainhibition of leukocyte migration involving MEKERK signal-ingrdquo BMCComplementary and AlternativeMedicine vol 13 no1 pp 45ndash55 2013

[7] C Yhirayha Formulation and skin penetration study of lyotropicliquid crystal incorporating mulberry stem (Morus alba L)extract [MS thesis] Naresuan University Phitsanulok Thai-land 2013

[8] M Pudla K Limposuwan and P Utaisincharoen ldquoBurkholde-ria pseudomallei-Induced expression of a negative regulatorsterile-120572 and Armadillo motif-containing protein in mousemacrophages a possible mechanism for suppression of theMyD88-independent pathwayrdquo Infection and Immunity vol 79no 7 pp 2921ndash2927 2011

[9] N Soonthornsit and T Pitaksuteepong ldquoMicroemulsion for-mulation containing mulberry stem extractrdquo in Proceedings ofthe 38th Congress on Science and Technology of Thailand 6pages Chiang Mai Thailand October 2012

[10] C H Hong S K Hur O-J Oh S S Kim K A Nam and S KLee ldquoEvaluation of natural products on inhibition of induciblecyclooxygenase (COX-2) and nitric oxide synthase (iNOS) incultured mouse macrophage cellsrdquo Journal of Ethnopharmacol-ogy vol 83 no 1-2 pp 153ndash159 2002

[11] E-M Choi and J-K Hwang ldquoEffects of Morus alba leafextract on the production of nitric oxide prostaglandin E2 andcytokines in RAW2647 macrophagesrdquo Fitoterapia vol 76 no7-8 pp 608ndash613 2005

[12] Z-G Yang K Matsuzaki S Takamatsu and S KitanakaldquoInhibitory effects of constituents from Morus alba var mul-ticaulis on Differentiation of 3T3-L1 cells and nitric Oxide

8 Evidence-Based Complementary and Alternative Medicine

Production in RAW2647 Cellsrdquo Molecules vol 16 no 7 pp6010ndash6022 2011

[13] H J Lim H-G Jin E-R Woo S K Lee and H P Kim ldquoTheroot barks ofMorus alba and the flavonoid constituents inhibitairway inflammationrdquo Journal of Ethnopharmacology vol 149no 1 pp 169ndash175 2013

[14] E Park S-M Lee J E Lee and J-H Kim ldquoAnti-inflammatoryactivity of mulberry leaf extract through inhibition of NF-120581BrdquoJournal of Functional Foods vol 5 no 1 pp 178ndash186 2013

[15] H Zelova Z Hanakova Z Cermakova et al ldquoEvaluation ofanti-inflammatory activity of prenylated substances isolatedfromMorus alba andMorus nigrardquo Journal of Natural Productsvol 77 no 6 pp 1297ndash1303 2014

[16] A Abate S Oberle and H Schroder ldquoLipopolysaccharide-induced expression of cyclooxygenase-2 inmousemacrophagesis inhibited by chloromethylketones and a direct inhibitor ofNF-120581B translocationrdquo Prostaglandins and Other Lipid Media-tors vol 56 no 5-6 pp 277ndash290 1998

[17] S-H Lee C-HKwak S-K Lee et al ldquoAnti-inflammatory effectof ascochlorin in lps-stimulated raw 2647 macrophage cells isaccompanied with the down-regulation of iNOS COX-2 andproinflammatory cytokines through NF-120581B ERK12 and p38signaling pathwayrdquo Journal of Cellular Biochemistry vol 117 no4 pp 978ndash987 2016

[18] L Parente ldquoPros and Cons of Selective Inhibition of COX-2versus Dual LipoxygenaseCOX Inhibition Is Two Better thanOnerdquo The Journal of Rheumatology vol 28 no 11 pp 2375ndash2382 2001

[19] U N Das ldquoCyclooxygenase (COX) lipoxygenase (LO) path-ways and generation of lipoxins resolvins protections andmaresinsrdquo in Molecular Basis of Health and Disease N DUndurti Ed vol 4 pp 59ndash61 Springer Science BusinessMediaBV Dordrecht Holland 2010

[20] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[21] C Riviere S Krisa L Pechamat et al ldquoPolyphenols from thestems of Morus alba and their inhibitory activity against nitricoxide production by lipopolysaccharide-activated microgliardquoFitoterapia vol 97 pp 253ndash260 2014

[22] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation and Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

minus +++++

20 40 10 (휇gml)5

MSE Oxyresveratrol

COX-2

β-Actin

LPS 100 ngml

(a)

000

030

060

090

120

150

Relat

ive e

xpre

ssio

n le

vels

of COX-2

minus +++++

20 40 10 (휇gml)5

lowastlowastlowast

lowastlowast lowastlowast

MSE Oxyresveratrol

LPS 100 ngml

(b)

Figure 6 The effects of MSE and oxyresveratrol on the expression of COX-2 mRNA in RAW 2647 cells The untreated cells were used as anegative control and the cells treated with only 100 ngml LPS served as a positive control (a) RT-PCR analysis for detection of COX-2mRNAexpression in LPS-stimulated RAW 2647 cells which were pretreated with MSE at the concentrations of 20 and 40120583gml or oxyresveratrolat the concentrations of 5 and 10120583gml for 6 h (b) The relative expression of COX-2 mRNA compared to 120573-actin The data were representedas means plusmn SD of three independent experiments The means marked with lowastlowast lowastlowastlowast are significantly different (119875 lt 0005) and (119875 lt 0001)respectively from that of the cells treated with only LPS

stems on LPS-induced RAW 2647 cells The investigationfound that concentrations at 20 and 40 120583gml had no toxiceffect The results suggest that the stem extract mediates itsanti-inflammatory activities through the inhibition of iNOSNO and COX-2 production Further studies are necessary tofully define the precise mechanisms involved

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors would like to acknowledge the financial supportfrom the yearly budget of Naresuan University Thailand(R2555B069 and R2558B053) the Center for Innovation inChemistry (PERCH-CIC) and National Research Council ofThailand Graduate Student Scholarship (2014) They expressspecial gratitude towards theQueen Sirikit SericultureCenter(Tak) for the supply of plant materials and to Mr CheinYhirayha for the purification of the oxyresveratrol Manythanks are due toMr RoyMorien of the Naresuan UniversityLanguage Centre for his editing assistance and advice onEnglish expression in this manuscript

References

[1] E W-C Chan P-Y Lye and S-K Wong ldquoPhytochemistrypharmacology and clinical trials of Morus albardquo Chinese Jour-nal of Natural Medicines vol 14 no 1 pp 17ndash30 2016

[2] K-O Chung B-Y Kim M-H Lee et al ldquoIn-vitro and in-vivoanti-inflammatory effect of oxyresveratrol fromMorus alba LrdquoJournal of Pharmacy and Pharmacology vol 55 no 12 pp 1695ndash1700 2003

[3] H S Lee D H Kim J E Hong J-Y Lee and E JKim ldquoOxyresveratrol suppresses lipopolysaccharide-induced

inflammatory responses in murine macrophagesrdquo Human andExperimental Toxicology vol 34 no 8 pp 808ndash818 2015

[4] P Thongsuk In vitro and clinical study of mulberry extractfor skin whitening product [MS thesis] Naresuan UniversityPhitsanulok Thailand 2007

[5] D Laveti M Kumar and R Hemalatha ldquoAnti-inflammatorytreatments for chronic diseases a reviewrdquo Inflammation ampAllergymdashDrug Targets vol 12 no 5 pp 349ndash361 2013

[6] Y-C Chen Y-J Tien C-H Chen et al ldquoMorus alba and activecompound oxyresveratrol exert anti-inflammatory activity viainhibition of leukocyte migration involving MEKERK signal-ingrdquo BMCComplementary and AlternativeMedicine vol 13 no1 pp 45ndash55 2013

[7] C Yhirayha Formulation and skin penetration study of lyotropicliquid crystal incorporating mulberry stem (Morus alba L)extract [MS thesis] Naresuan University Phitsanulok Thai-land 2013

[8] M Pudla K Limposuwan and P Utaisincharoen ldquoBurkholde-ria pseudomallei-Induced expression of a negative regulatorsterile-120572 and Armadillo motif-containing protein in mousemacrophages a possible mechanism for suppression of theMyD88-independent pathwayrdquo Infection and Immunity vol 79no 7 pp 2921ndash2927 2011

[9] N Soonthornsit and T Pitaksuteepong ldquoMicroemulsion for-mulation containing mulberry stem extractrdquo in Proceedings ofthe 38th Congress on Science and Technology of Thailand 6pages Chiang Mai Thailand October 2012

[10] C H Hong S K Hur O-J Oh S S Kim K A Nam and S KLee ldquoEvaluation of natural products on inhibition of induciblecyclooxygenase (COX-2) and nitric oxide synthase (iNOS) incultured mouse macrophage cellsrdquo Journal of Ethnopharmacol-ogy vol 83 no 1-2 pp 153ndash159 2002

[11] E-M Choi and J-K Hwang ldquoEffects of Morus alba leafextract on the production of nitric oxide prostaglandin E2 andcytokines in RAW2647 macrophagesrdquo Fitoterapia vol 76 no7-8 pp 608ndash613 2005

[12] Z-G Yang K Matsuzaki S Takamatsu and S KitanakaldquoInhibitory effects of constituents from Morus alba var mul-ticaulis on Differentiation of 3T3-L1 cells and nitric Oxide

8 Evidence-Based Complementary and Alternative Medicine

Production in RAW2647 Cellsrdquo Molecules vol 16 no 7 pp6010ndash6022 2011

[13] H J Lim H-G Jin E-R Woo S K Lee and H P Kim ldquoTheroot barks ofMorus alba and the flavonoid constituents inhibitairway inflammationrdquo Journal of Ethnopharmacology vol 149no 1 pp 169ndash175 2013

[14] E Park S-M Lee J E Lee and J-H Kim ldquoAnti-inflammatoryactivity of mulberry leaf extract through inhibition of NF-120581BrdquoJournal of Functional Foods vol 5 no 1 pp 178ndash186 2013

[15] H Zelova Z Hanakova Z Cermakova et al ldquoEvaluation ofanti-inflammatory activity of prenylated substances isolatedfromMorus alba andMorus nigrardquo Journal of Natural Productsvol 77 no 6 pp 1297ndash1303 2014

[16] A Abate S Oberle and H Schroder ldquoLipopolysaccharide-induced expression of cyclooxygenase-2 inmousemacrophagesis inhibited by chloromethylketones and a direct inhibitor ofNF-120581B translocationrdquo Prostaglandins and Other Lipid Media-tors vol 56 no 5-6 pp 277ndash290 1998

[17] S-H Lee C-HKwak S-K Lee et al ldquoAnti-inflammatory effectof ascochlorin in lps-stimulated raw 2647 macrophage cells isaccompanied with the down-regulation of iNOS COX-2 andproinflammatory cytokines through NF-120581B ERK12 and p38signaling pathwayrdquo Journal of Cellular Biochemistry vol 117 no4 pp 978ndash987 2016

[18] L Parente ldquoPros and Cons of Selective Inhibition of COX-2versus Dual LipoxygenaseCOX Inhibition Is Two Better thanOnerdquo The Journal of Rheumatology vol 28 no 11 pp 2375ndash2382 2001

[19] U N Das ldquoCyclooxygenase (COX) lipoxygenase (LO) path-ways and generation of lipoxins resolvins protections andmaresinsrdquo in Molecular Basis of Health and Disease N DUndurti Ed vol 4 pp 59ndash61 Springer Science BusinessMediaBV Dordrecht Holland 2010

[20] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[21] C Riviere S Krisa L Pechamat et al ldquoPolyphenols from thestems of Morus alba and their inhibitory activity against nitricoxide production by lipopolysaccharide-activated microgliardquoFitoterapia vol 97 pp 253ndash260 2014

[22] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation and Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Production in RAW2647 Cellsrdquo Molecules vol 16 no 7 pp6010ndash6022 2011

[13] H J Lim H-G Jin E-R Woo S K Lee and H P Kim ldquoTheroot barks ofMorus alba and the flavonoid constituents inhibitairway inflammationrdquo Journal of Ethnopharmacology vol 149no 1 pp 169ndash175 2013

[14] E Park S-M Lee J E Lee and J-H Kim ldquoAnti-inflammatoryactivity of mulberry leaf extract through inhibition of NF-120581BrdquoJournal of Functional Foods vol 5 no 1 pp 178ndash186 2013

[15] H Zelova Z Hanakova Z Cermakova et al ldquoEvaluation ofanti-inflammatory activity of prenylated substances isolatedfromMorus alba andMorus nigrardquo Journal of Natural Productsvol 77 no 6 pp 1297ndash1303 2014

[16] A Abate S Oberle and H Schroder ldquoLipopolysaccharide-induced expression of cyclooxygenase-2 inmousemacrophagesis inhibited by chloromethylketones and a direct inhibitor ofNF-120581B translocationrdquo Prostaglandins and Other Lipid Media-tors vol 56 no 5-6 pp 277ndash290 1998

[17] S-H Lee C-HKwak S-K Lee et al ldquoAnti-inflammatory effectof ascochlorin in lps-stimulated raw 2647 macrophage cells isaccompanied with the down-regulation of iNOS COX-2 andproinflammatory cytokines through NF-120581B ERK12 and p38signaling pathwayrdquo Journal of Cellular Biochemistry vol 117 no4 pp 978ndash987 2016

[18] L Parente ldquoPros and Cons of Selective Inhibition of COX-2versus Dual LipoxygenaseCOX Inhibition Is Two Better thanOnerdquo The Journal of Rheumatology vol 28 no 11 pp 2375ndash2382 2001

[19] U N Das ldquoCyclooxygenase (COX) lipoxygenase (LO) path-ways and generation of lipoxins resolvins protections andmaresinsrdquo in Molecular Basis of Health and Disease N DUndurti Ed vol 4 pp 59ndash61 Springer Science BusinessMediaBV Dordrecht Holland 2010

[20] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[21] C Riviere S Krisa L Pechamat et al ldquoPolyphenols from thestems of Morus alba and their inhibitory activity against nitricoxide production by lipopolysaccharide-activated microgliardquoFitoterapia vol 97 pp 253ndash260 2014

[22] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation and Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom