research article assessment of cytotoxicity, antioxidant ...extracts against human caucasian skin...

Research ArticleIn Vitro Assessment of Cytotoxicity Antioxidantand Anti-Inflammatory Activities of Ricinus communis(Euphorbiaceae) Leaf Extracts

Vhutshilo Nemudzivhadi and Peter Masoko

Department of Biochemistry Microbiology and Biotechnology University of Limpopo Turfloop CampusPrivate Bag X1106 Sovenga 0727 South Africa

Correspondence should be addressed to Peter Masoko petermasokoulacza

Received 6 August 2014 Revised 22 October 2014 Accepted 28 October 2014 Published 16 November 2014

Academic Editor William C S Cho

Copyright copy 2014 V Nemudzivhadi and P Masoko This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Ricinus communis has been utilized traditionally as medicine to treat inflammatory related diseases including wounds soresand boils The leaves of R communis were sequentially extracted with n-hexane dichloromethane acetone and methanol usingserial exhaustive extraction method Antioxidant activity of all crude extracts was quantitatively measured against 221015840-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) free radical molecules using ABTS+ assay Cytotoxic effect and anti-inflammatoryactivity of R communis leaves extracts were evaluated on Human Caucasian skin fibroblast and Raw 2647 macrophage cell linesrespectively Methanol extract had the highest percentage free radical (ABTS+) scavenging activity of 95 at 250mgmL acetone91 dichloromethane 62 and hexane the least (50) Percentage scavenging activity of ABTS+ free radical molecules increaseswith increase in concentrations of the plant extracts Hexane and dichloromethane extracts had more than 90 cell viabilityat 100 120583gmL after 24 and 48 hours of exposure Methanol extract had LC

50of 784 120583gmL after 24-hour exposure hexane had

6293 120583gmL and dichloromethane 5736 120583gmL and 5446 120583gmL was the lowest with acetone extract The study present the firstreport on the scavenging activity of R communis leaf extracts against ABTS+ radicals and cytotoxic effects on human Caucasianskin fibroblast cell lines

1 Introduction

Since ancient times medicinal plants have played a vital rolein preserving human health The use of medicinal plants hasincreased in rural areas and developing countriesThis is dueto lack of hospitals poverty and an increased demand ofinexpensive medicines [1] Medicinal plants continue to pro-vide humanity with new remedies It is therefore importantto explore medicinal plants for their safety quality toxicityappropriate amount of plant materials to use and efficacyNatural products of plants possess several biological activitiesincluding antioxidant and anti-inflammatory activity [2 3]Medicinal plants are rich in active phytochemical compoundswith various biological activities Researchers are highlyinterested in studying plants with the aim of isolating novelactive drugs to replace synthetic drugs present in the market

The availability of these plants constituents provides a sourceof natural drugs for modern medicine [4] Hence cytotoxiclevel of medicinal plants must also be evaluated against hostcells The safety of plants as a potential therapeutically agentsmust be ascertained and the side effects should be acceptableto the host Bioactive compounds with no or less toxic effectto the host are the good candidates for formulation of drugs[5]

Antioxidants play an important role in neutralizingfree radical species which are produced as end or by-products of normal biochemical reactions in normal sys-tem [6] High amounts of free radical molecules causeoxidative stress in cells which result in damaging essen-tial macromolecules including DNA lipids and proteinsThe damage of macromolecules leads to inflammation andmany degenerative conditions such as Parkinsonrsquos diseases

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 625961 8 pageshttpdxdoiorg1011552014625961

2 Evidence-Based Complementary and Alternative Medicine

atherosclerosis aging immunosuppression ischemic heartdisease diabetes hair loss membrane lipid peroxidationand decreased membrane fluidity [3 6ndash8] Reactive oxygenspecies are also reported as carcinogenic and mutagenicagents [9] Signs of inflammation are swollen joints jointpain stiffness and loss of joint functions Nonsteroidalanti-inflammatory drug (NSAIDs) such as ibuprofen andnaproxen are anti-inflammatory drugs currently used fortreatment of inflammation These drugs are known to causesevere side effects in the body such as heart attacks and stroke[10]

Plant constituents are responsible for both free radi-cals scavenging and anti-inflammatory activity Secondarymetabolites are responsible for biological activities of plantsincluding terpenoids [11] phenolic compounds (flavonoidsphenolic acids quinones coumarins lignans stilbenes tan-nins) and nitrogen compounds (alkaloids amines andbetalains) and carotenoids [12] Iqbal et alrsquos [13] reports areavailable which suggest that phenolic compounds are thepotent antioxidant compounds from medicinal plants

Ricinus communis (Castor oil plant) belongs to the familyEuphorbiaceae Castor oil plant is originally from Africa andis now found in all tropical countries [14] Leaves barksseeds roots and oil of the plant have been traditionally usedfor many purposes throughout the world The leaf root andseed oil of the plant have also been used for treatment ofinflammation and liver disorders in India [15] In TunisiaR communis is used as a contraceptive herbal drug fortreatment of cold and tumors as a hypoglycemic and as alaxative R communis is well known for its biological activi-ties most important of which are hepatoprotective laxativeantidiabetic and antifertility activities [16] Literature searchrevealed that essential oils from R communis have beenreported to have potential antimicrobial anticarcinogenicantioxidant and antidiabetic activities [17ndash19]The aimof thisstudy was to evaluate the cytotoxic effects of R communisextracts against Human Caucasian skin fibroblast (BUD-8)cell line as well as its antioxidant activityusing 221015840-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) freeradicals and anti-inflammation activity on the Raw 2647macrophages cell line

2 Methods and Materials

21 Plant Collection and Processing Leaves were collectedin summer of 2011 from University of Limpopo (Turfloopcampus) South Africa Voucher specimens in the gardenHerbarium and tree labels verified the identity of the plantPlant was confirmed by Dr Brownyn Egan (Herbarium) andthe accession detail was UNIN 11352 The plant was selectedbased on the high antioxidant activity demonstrated inprevious in vitro study [20] Leaves were separated from twigsand dried at room temperature The dried plant materialswere milled to fine powder and stored at room temperaturein closed bottles in the dark until use for the extraction

22 Extraction Procedure Dried plant materials weresequentially extracted by mixing 10 g of plant material with

100mL of hexane dichloromethane acetone and methanolin glass bottle Bottles were vigorously shaken at a speed of300 rpm overnight Plant residues were allowed to settleand the supernatant was filtered and evaporated usinga rotary evaporator (BUCHi Labotec rotavapor modelR-205 Germany) and transferred into preweighed labeledglass beakers The process was repeated three times toexhaustively extract the plant material and the extracts foreach solvent were combinedThe solvent was removed undera stream of cold air at room temperature Plant extractswere reconstituted using acetone to a final concentration of10mgmL

23 Analysis of Extracts by TLC Ten microliters of10mgmL plant extracts were loaded on a thin layerchromatography plates Three separation systems of varyingpolarities were used to analyse plant extracts by thinlayer chromatography (TLC) (Fluka silica gel F

254plates)

benzene ethanol ammonium hydroxide (BEA) (36 4 04)chloroform ethyl acetate formic acid (CEF) (20 16 4)and ethyl acetate methanol water (EMW) (20 108 8)Chromatograms were examined under ultraviolet light (254and 365 nm) and sprayed with freshly prepared vanillin sprayreagent (01 g vanillin 28mL methanol and 1mL sulphuricacid) to visualise separated compounds The plates werecarefully heated at 110∘C for optimal colour development[21]

24 Antioxidant Activities

241 Qualitative 22-Diphenyl-1-picrylhydrazyl Assay on TLCThin layer chromatography plates were used to separate crudeextracts as described in Section 23 To detect antioxidantactivity chromatograms were sprayed with 02 (wv) 22-diphenyl-1-picrylhydrazyl (DPPH) (Sigma) in methanol asan indicator Yellow zones appearing on the chromatogramsindicate the presence of scavenging activity of free radicals bycompounds presence in plant extracts [22]

242 Quantitative Antioxidant Assay Using ABTS+ A stocksolution of 7mM of ABTS+ was prepared according to vanden Berg et al [23] by mixing 8mg of ABTS with 1mL ofwater ABTS free radicals were produced by reacting equalvolume of ABTS stock solution with 132mg in 10mL of(245mM) potassium sulphate The solution was preparedand incubated for 12ndash16 hours in the dark at room tempera-ture until the reaction was complete and stored at 4∘C untiluse Serial dilution method was used to prepare differentconcentrations of crude extracts (hexane dichloromethaneacetone and methanol) ABTS+ was diluted with waterfor equilibration of absorbance 070 (plusmn002) at 734 nm andappropriate blank was used without test samples Hundredmicroliters of ABTS+ was added to 100120583L of 250 125 063016 008 and 004mgmL concentrations of crude extractsThe absorbance reading was taken after 6 minutes following

Evidence-Based Complementary and Alternative Medicine 3

BEA

CEF

EMW

Hexane DCM Acetone Methanol

I II III I II III I II III I II III


(a)

Dichloromethane

BEA

CEF

EMW

Hexane Acetone Methanol



(b)

Figure 1 Chromatograms of R communis leaves extracted with hexane dichloromethane (DCM) acetone and methanol in lane from leftto right separated with three solvent systems BEA CEF and EMW from top to bottom and sprayed with vanillin-sulphuric acid reagent (a)and 02 DPPH in methanol as an indicator (b) yellow spots against purple background indicate antioxidant activity

the reaction All determinations were carried out in triplicateand calculated as follows

Total antioxidant activity

=Absorbancecontrol minus Absorbancesample

Absorbancecontroltimes 100

(1)

25 In Vitro Cytotoxicity Using MTT Assay Cell viabilitywas assessed according to Mosaddegh et al [24] on theHuman Caucasian skin fibroblast (Bud-8) cell line Theproliferation rates of Bud-8 cell line after treatment with plantextracts were determined by the 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) assay MTTis reduced by mitochondrial dehydrogenases to the water-insoluble pink compound formazan depending on theviability of cells Cells were maintained in the DulbeccorsquosModified Essential Medium (DMEM) supplemented with10 fetal bovine serum (FBS) and 1x Penicillin-Streptomycin-Neomycin (PSN) Hundred microliters of cells (4 times 104cellsmL) was seeded in 96-well plates and incubated at 37∘C5 carbon dioxide for 24 hours After 24 hours of incubationthe cells were treated with 100 120583L of 100 200 300 400

and 500 120583gmL of plant extracts (hexane dichloromethaneacetone and methanol) Plates were incubated at 37∘C 5CO2for 24 and 48 hours After incubation morphology of

cells was examined under microscope Twenty microliters ofMTT solution (5mgmL) (Sigma) was added to each wellThe plates were further incubated for 2 to 4 hours and themediumwas removed Formazan crystals were dissolvedwith100 120583L of dimethyl sulfoxide (DMSO) The absorbance wasmeasured at 560 nm and percentages of cell viability and LC

50

of cells were calculated

cell viability =Absorbancesample

Absorbancecontroltimes 100 (2)

whereAbsorbancecontrol is the absorbance of cells treatedwithDMSO 1 and Absorbancesample is the absorbance of cellstreated with test sample

26 Anti-Inflammatory Activity Using PhagoBurst AssayTwo hundred microliters of cells (Raw 2647 macrophages)was transferred to a coverslip in a multiwell plate The cellswere incubated at 37∘C 5 CO

2overnight to allow cells

to attach Cells were treated with 100 120583L of plant extracts


(100 120583gmL of hexane dichloromethane acetone andmethanol) and 10mgmLof LPS for 24 hours Twomicrolitersof PMA was also added for 30 minutes After incubationthe medium was aspirated Cells were stained with 50 120583Lof 10minus5M of H

2DCF-DA and incubated for 20 minutes

in the dark Cells were stained for the second time with50120583L of 20120583gmL 410158406-diamidine-2-phenylindole (DAPI)and further incubated for 20 minutes in the dark Stainingsolutions were removed and 37 paraformaldehyde wasadded to a coverslip to fix the cells Coverslips were mountedto a microscope slide and examined under fluorescencemicroscope

3 Results and Discussion

Medicinal plants have unlimited capacity to synthesis ofbioactive compounds that are effective and have fewer sideeffects compared to synthetic drugs Bioactive compoundsfrom plants have shown over the years to have various bio-logical activities Scientists have developed a greater interestof using these compounds in formulation of new and noveldrugs because of their biological activities and reliability [25]Crude extracts were separated with three mobile phases andsprayed with vanillin-sulphuric acid to visualize phytochem-ical compounds present (Figure 1(a)) To qualitatively analysecrude extracts for antioxidant activity chromatograms weresprayed with 02 DPPH with yellowish spots indicating thepresence of antioxidant compounds (Figure 1(b)) Acetoneextracts revealed strong antioxidant compounds against 02DPPH free radicals in EMW solvent system while hexaneDCM and methanol extracts did not Acetone and methanolextracts showed antioxidant compounds in CEF and EMWsolvent systems even though active compounds were notseparated due to their polarity relative to the solvent systemsIqbal et al [13] also reported on the potent antioxidant activ-ity of the aerial part of R communis So far there is nothingreported on the scavenging activity of R communis leafextracts against ABTS free radicals Antioxidant activity ofhexane dichloromethane acetone and methanol extracts ofR communiswas also quantified usingABTS+ decolonizationmethod Addition of antioxidants to free radicals reducedthe blue chromophores ABTS+ to colorless Methanol extracthad the highest percentage free radical (ABTS+) scavengingactivity of 95 at 250mgmL acetone 91 dichloromethane62 andhexane 50 the least (Figure 2) Polar solvents seemto extract compounds responsible for antioxidant activityof R communis ABTS+ decolorization method is used forthe screening of antioxidant activity of plant extracts andis applicable to both lipophilic and hydrophilic antioxidants[26] Percentage scavenging activity of both ABTS andDPPH free radical molecules increases with the increase ofconcentrations of the extracts The present study suggeststhe leaves of R communis as good source of antioxidantcompounds to scavenge both ABTS and DPPH free radicalsScavenging activity of ABTS free radical molecules has neverbeen reported before on the leaves ofR communis although ithas previously been reported in the seeds of the R communisby Surveswaran et al [27]

0

20

40

60

80

100

120

004 008 016 031 063 125 250

Scav

engi

ng ac

tivity

()

Concentrations (mgmL)

HexaneDCM

AcetoneMethanol

Figure 2 Percentage scavenging activity of ABTS+ free radicals withrespect to increasing concentration of R communis crude extractshexane dichloromethane (DCM) acetone and methanol extracts

There are other methods which are used to test forantioxidants like ferric reducing antioxidant power (FRAP)method electron spin resonance spectrometry and Phyco-erythrin assay FRAP method is based on the reductionof a ferroin analog the Fe3+ complex of tripyridyltriazineFe(TPTZ)3+ to the intensely blue coloured Fe2+ complexFe(TPTZ)2+ by antioxidants in acidic medium Results areobtained as absorbance increases at 593 nm and can beexpressed as micromolar Fe2+ equivalents or relative to anantioxidant standard This method is automated howeverthe measured reducing capacity does not necessarily reflectantioxidant activity It provides instead a very useful ldquototalrdquoantioxidant concentration without measurement and sum-mation of the concentration of all antioxidants involved

Electron spin resonance (ESR) spectrometry is the onlyanalytical technique that can specifically detect the freeradicals involved in autoxidation and related processes Theassay is intrinsically sensitive to stable free radicals such asdi-tert-butyl nitroxide ESR is unfortunately insensitive todetecting the reactive short-lived free radicals involved inautoxidation (lifetimes vary from 10-9 s for the hydroxyl rad-ical to several seconds for the peroxyl radical) Phycoerythrinassay is particularly useful in screening for compounds thatprotect against damage by chelating metal ions necessary forsite-specific formation of the radical species The inhibitionof oxidation by an antioxidant can be examined by theretardation of the loss of fluorescence with the inhibitionbeing proportional to the antioxidant activity Results can becalculated using the differences in areas under the phycoery-thrin decay curves between the blank and a sample and areexpressed in Trolox equivalents

Amajor concern about bioactive compounds from plantsis that some of these compounds are toxic to our nor-mal system therefore safety is critical in development ofnovel drugs [5] In this study cytotoxic effect of hexanedichloromethane acetone and methanol extracts of R com-munis at concentration ranging from 100ndash500120583gmL was


0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol

Concentrations (120583gmL)

Cel

l via

bilit

y (

)24hrs

(a)

0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol

Cel

l via

bilit

y (

)

48hrs


(b)

Figure 3 (a) Cytotoxicity effect of R communis leaf extracts against Bud-8 cell line after 24 hours of exposure using MTT assay at theconcentration ranging from 100 to 500 120583gmL (b) Cytotoxicity effect of R communis leaf extracts against Bud-8 cell line after 48 hours ofexposure using MTT assay at the concentration ranging from 100 to 500120583gmL

investigated onHumanCaucasian skin fibroblast (Bud-8) cellline by using MTT assay Effect of R communis extracts onBud-8 cell line was evaluated after 24 (Figure 3(a)) and 48hours (Figure 3(b)) of cell exposure to the extracts Percentagecell viability was calculated by measuring the absorbanceof pink colour formazan formed from reduction of MTTsolution by the presence of mitochondrial dehydrogenase inviable cells All crude extracts were toxic to Bud-8 cell line atthe higher concentration of 500120583gmL Morphology of cellswas also altered from its normal shape of fibroblast to ovalshape because of the toxic effect of R communis extracts tothe cells However at the lower concentrations (100120583gmL)hexane and dichloromethane extracts had more than 90cell viability after 24 and 48 hours of exposure Acetoneand methanol extracts had less than 90 cell viability Cellviability decreased with the increase in concentrations ofplant extracts Higher concentrations ofR communis extractswere observed to be toxic on Bud-8 cell line Rana et al [28]reported the toxic effect of ricin isolated from the seeds of Rcommunis Ricin kills the cells by disrupting protein synthesisin the cells Ricin was the only compound isolated from Rcommunis seeds that was reported to have high toxic effectonmammalian cells Leaves ofR communis also contain ricinbut in lower concentration The present finding presents thefirst report on the cytotoxicity effects of R communis leafextracts against Human Caucasian skin fibroblast cell lines

The LC50of R communis extracts on Bud-8 cell line was

also calculated for each extract after 24 and 48 hours ofexposure (Table 1) LC

50indicates the lowest concentration of

plant extracts that inhibits 50 of cells Methanol extract hadhigh LC

50of 784120583gmL after 24-hour exposure followed by

hexane (6293120583gmL) and dichloromethane (5736120583gmL)and the lowest was in acetone extract (5446 120583gmL) Morobeet al [5] also reported low toxic effect of methanol extracts onMAGI CCR5+ cell lines after 24 hoursThe LC

50for all crude

extracts decreased after 48 hours of exposure Hexane had thehighest LC

50of 495 120583gmL andmethanol had the lowest LC

50

Table 1 The LC50 of R communis extracts on BUD-8 cell lines after24 and 48 hours of exposure

Extracts LC50 (120583gmL)24 hours 48 hours

Hexane 6293 495Dichloromethane 5736 467Acetone 5445 471Methanol 784 3871

of 3871 120583gmL In general low LC50

values represent hightoxicity Extracts with high LC

50are preferable to work with

because of their lower toxicity effects on the host cells Thecytotoxicity of the oil of R communis was also reported withLC50values less than 263mgmL for HeLa cell line [19] The

CC50values of R communis leaf extracts on Vero cell line was

reported at 165mgmL [29]In Cell Proliferation Reagent WST-1 assay the Cell Pro-

liferation Reagent WST-1 is a ready-to-use colorimetric assayfor the nonradioactive quantification of cellular proliferationviability and cytotoxicity Sample material is either adherentor suspension cells cultured in 96-well microplatesThe assayis based in the enzymatic cleavage of the tetrazolium saltWST-1 to formazan by cellular mitochondrial dehydroge-nases present in viable cells In principle WST-1 works sim-ilarly to MTT by reacting with the mitochondrial succinate-tetrazolium reductase forming the formazan dye TheWST-1reagent produces a water-soluble formazan rather than thewater-insoluble product of the MTT assay It can be usedfor measurement of cell proliferation in response to growthfactors cytokines mitogens and nutrients and analysis ofcytotoxic and cytostatic compounds such as anticancer drugsand other pharmaceutical compounds

Roots extracts of R communis have been reported toneutralize free radical molecules and anti-inflammatoryactivityThe same study suggested that the anti-inflammatory


Control LPS PMA Hexane LPS PMA

Dichloromethane LPS PMA Acetone LPS PMA

Methanol LPS PMA

Figure 4 Effect of R communis extracts on Raw 2647 macrophages cells in response to oxidative stress and inflammation Reactive oxygenspecies were induced by LPS and PMA stimulants Cells were fixed and stained with H

2DCF-DA to measure the amount of free radical

molecules produced in the cells (green) and DAPI to locate the nucleus in the cells (blue)

activity of roots extracts of R communis was because of thepresence of flavonoids [30] The fluorogenic cell permeantH2DCF-DA (green cells) was used to measure the level

of reactive oxygen species (ROS) in activated Raw 2647macrophage cells induced by LPS and PMA stimulants(Figure 4) High level of reactive oxygen species tends toattack macromolecules and this facilitates cells to undergooxidative stress and inflammatory response Antioxidantcompounds scavenge the free radical molecules by donating

one electron or proton to a molecule High intensity ofgreen colour is the indication of high level of free radicalmoleculeswhich can lead cells to undergo oxidative stress andinflammation Reactive oxygen species act as a mediator toregulate cytokines production through activation of the tran-scription factors such as NF-120581BThis suggests the direct linkbetween ROS and other cytokines to initiate inflammatoryresponse [31] When dihydrodichlorofluorescein diacetate(H2DCF-DA) penetrates through the plasma membrane of


cells and is then deesterified to a hydrophilic alcoholdihydrodichlorofluorescein (H

2DCF) it is oxidized to fluo-

rochrome DCF (27-dichlorofluorescein) by the presence ofreactive oxygen species that fluoresce green when excitedwith blue light The brightness of DCF fluorescence reflectsthe level at which reactive oxygen species are presentin the cell [32] A reduced internal H

2DCF fluorescence

intensity is an indication of reduced level of free radi-cal molecules by antioxidant compounds extracted fromR communis leaves Combination of hexane and acetoneextracts with PMA and LPS stimulants depicts low fluo-rescence intensity as compared to PMA and LPS stimu-lated cells Hexane acetone and methanol extracts wereobserved to reduce the level of ROS formation in cellswhile dichloromethane extract did not Methanol extractsof R communis have been reported to contain flavonoid aconstituent that has been reported as the anti-inflammatoryagent of medicinal plants [33] The root extracts of R com-munis have been reported for their strong anti-inflammatorycompounds [30] The possibility that active constituentpresent in the roots may be responsible for observedactivity in the leaves may not be ruled out Roots andleaves materials may contain similar constituents at varyingconcentrations

The Griess assay was not considered because of itslimitations This assay can only be used to measure thenitric oxide excluding other free radical molecules In Griessassay nitric oxide (NO) is a molecular mediator of manyphysiological processes including inflammation Griess assayis a colorimetric assay to measure the levels of nitriteoxide in aqueous solution Griess assay involves the useof the Griess diazotization reaction to spectrometricallydetect nitrite formed by the spontaneous oxidation of NOunder physiological conditions The Griess reaction can alsobe used to analyze nitrate via its catalytic reduction tonitrite

4 Conclusion

The leaves ofR communis revealed strong antioxidant activityin both assay tests Although some studies have reported onthe cytotoxicity effects and anti-inflammatory activity of Rcommunis leaf extracts on other cell lines this is the first studyto report on the cytotoxicity effect and anti-inflammatoryactivity of R communis leaves on Human Caucasian skinfibroblast and Raw 2647 macrophage cell lines respectivelyCrude extracts of R communis leaves revealed low toxiceffect on Bud-8 cell line at lower concentrations while athigh concentrations the extracts are toxic The present studyalso agrees with previous findings indicating the potent anti-inflammatory activity of R communis using other plantsparts The study serves as a scientific proof for the use ofR communis leaves in traditional medicine for treatment ofinflammatory related diseases Further studies are requiredto isolate anti-inflammatory compounds Traditional healerswill be advised to use the leaves instead of uprooting theplants

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors would like to acknowledge National ResearchFoundation (NRF) for financial support

References

[1] L J Shai L JMcGaw PMasoko and JN Eloff ldquoAntifungal andantibacterial activity of seven traditionally used South Africanplant species active against Candida albicansrdquo South AfricanJournal of Botany vol 74 no 4 pp 677ndash684 2008

[2] N Huda-Faujan A Noriham A S Norrakiah and A S BabjildquoAntioxidant activity of plants methanolic extracts containingphenolic compoundsrdquo African Journal of Biotechnology vol 8no 3 pp 484ndash489 2009

[3] V R Patel P P Patel and S S Kujal ldquoAntioxidant activity ofsome selected medicinal plants in western Region of IndiardquoAdvances in Biological Research vol 4 no 1 pp 23ndash26 2010

[4] W H Talib and A M Mahasneh ldquoAntiproliferative activityof plant extracts used against cancer in traditional medicinerdquoScientia Pharmaceutica vol 78 no 1 pp 33ndash45 2010

[5] I C Morobe N S Mthethwa M A Bisi-Johnson et al ldquoCyto-toxic effects and safety profiles of extracts of active medicinalplants from South Africardquo Journal ofMicrobiology Research vol2 no 6 pp 176ndash182 2012

[6] M Jimenez-Estrada C Velazquez-Contreras A Garibay-Escobar et al ldquoIn vitro antioxidant and antiproliferative activ-ities of plants of the ethnopharmacopeia from northwest ofMexicordquoBMCComplementary andAlternativeMedicine vol 13article 12 2013

[7] M Kratchanova P Denev M Ciz A Lojek and A MihailovldquoEvaluation of antioxidant activity of medicinal plants con-taining polyphenol compounds Comparison of two extractionsystemsrdquo Acta Biochimica Polonica vol 57 no 2 pp 229ndash2342010

[8] S Magama M I Lieta and A O Asita ldquoAntioxidant andfree radical scavenging properties of four plant species usedin traditional medicine in Lesothordquo International Journal ofMedicinal Plant Research vol 2 no 3 pp 170ndash178 2013

[9] A Aguirre and R Borneo ldquoAntioxidant effects of four nativemedicinal plants collected in Cordobardquo Molecular MedicinalChemistry vol 12 pp 1ndash3 2010

[10] D A Kumar C Kshitij and K Nishteswar ldquoRicinus communisLinnmdashan Ayurveda and research perspectivesrdquo InternationalJournal of Universal Pharmacy and Life Sciences vol 3 no 3pp 71ndash90 2013

[11] P Masoko and J N Eloff ldquoScreening of twenty-four SouthAfricanCombretum and six Terminalia species (Combretaceae)for antioxidant activitesrdquo African Journal of Traditional Com-plementary and Alternative Medicines vol 4 no 2 pp 231ndash2392007

[12] M Bichra C El-Modafar A El-Abbassi H Bouamama andF Benkhalti ldquoAntioxidant activities and phenolic profile of sixMoroccan selected herbsrdquo Journal of Microbiology Biotechnol-ogy and Food Sciences vol 2 no 4 pp 2320ndash2338 2013


[13] J Iqbal S Zaib U Farooq A Khan I Bibi and S SulemanldquoAntioxidant Antimicrobial and free radical scavenging poten-tial of aerial parts of Periploca aphylla and Ricinus communisrdquoISRN Pharmacology vol 2012 Article ID 563267 6 pages 2012

[14] E Christy Jeyaseelan and P T Justin Jashothan ldquoIn vitro controlof Staphylococcus aureus (NCTC 6571) and Escherichia coli(ATCC 25922) by Ricinus communis Lrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 9 pp 717ndash721 2012

[15] D J Taur M G Waghmare R S Bandal and R Y PatilldquoAntinociceptive activity of Ricinus communis L leavesrdquo AsianPacific Journal of Tropical Biomedicine vol 1 no 2 pp 139ndash1412011

[16] D Rekha S Tamil-Selvi R Bharathidasan A PanneerselvamR Ilakkiya andR Jayapal ldquoStudy ofmedicinal plants used fromkoothanoallur and marakkadai Thiruvarur district of Tamilnadu IndiardquoHygeia Journal for Drugs and Medicines vol 5 no1 pp 164ndash170 2013

[17] P Shokeen P Anand Y KMurali andV Tandon ldquoAntidiabeticactivity of 50 ethanolic extract of Ricinus communis and itspurified fractionsrdquo Food and Chemical Toxicology vol 46 no11 pp 3458ndash3466 2008

[18] A Kadri N Gharsallah M Damak and R Gdoura ldquoChemicalcomposition and in vitro antioxidant properties of essential oilof Ricinus communis Lrdquo Journal of Medicinal Plants Researchvol 5 no 8 pp 1466ndash1470 2011

[19] Z Zarai I B Chobba R B Mansour A Bekir N Gharsallahand A Kadri ldquoEssential oil of the leaves of Ricinus communisL in vitro cytotoxicity and antimicrobial propertiesrdquo Lipids inHealth and Disease vol 11 article 102 2012

[20] V Nemudzivhadi Isolation and characterization of antibacterialand antioxidant compounds from Ricinus communis leaves [MSthesis] University of Limpopo (Turfloop campus) LimpopoSouth Africa 2014

[21] MKotze and JN Eloff ldquoExtraction of antibacterial compoundsfromCombretummicrophyllum (Combretaceae)rdquo SouthAfricanJournal of Botany vol 68 no 1 pp 62ndash67 2002

[22] C Deby and G Magotteaux ldquoRelationship between essentialfatty acids and tissue antioxidant levels in micerdquo ComptesRendus des Seances de la Societe de Biologie et de Ses Filiales vol164 no 12 pp 2675ndash2681 1970

[23] R van den Berg G R M M Haenen H van den Bergand A Bast ldquoApplicability of an improved Trolox equivalentantioxidant capacity (TEAC) assay for evaluation of antioxidantcapacity measurements of mixturesrdquo Food Chemistry vol 66no 4 pp 511ndash517 1999

[24] M Mosaddegh S Esmaeili F Naghibi et al ldquoEthnomedicalsurvey and cytotoxic activity of medicinal plant extracts usedin Kohgiluyeh and Boyerahmad Province in Iranrdquo Journal ofHerbs Spices and Medicinal Plants vol 18 no 3 pp 211ndash2212012

[25] S Vijayarathna and S Sasidharan ldquoCytotoxicity of methanolextracts of Elaeis guineensis on MCF-7 and Vero cell linesrdquoAsian Pacific Journal of Tropical Biomedicine vol 2 no 10 pp826ndash829 2012

[26] R Rajamurugan V Deepa M Sivashanmugam and CM Raghavan ldquoPhytochemistry antioxidant and antibacterialactivities of medicinal plantsmdasha comparative studyrdquo Interna-tional Journal of Current Research and Review vol 5 no 2 pp8ndash19 2013

[27] S Surveswaran Y-Z Cai H Corke and M Sun ldquoSystematicevaluation of natural phenolic antioxidants from 133 Indian

medicinal plantsrdquo Food Chemistry vol 102 no 3 pp 938ndash9532007

[28] M Rana H Dhamija B Prashar and S Sharma ldquoRicinuscommunis Lmdasha reviewrdquo International Journal of PharmTechResearch vol 4 no 4 pp 1706ndash1711 2012

[29] A N B Salem R Zyed L M Ali S Nidhal S Souad and AMahjoub ldquoCytotoxic effect of nanoparticles synthesized fromSalvia officinalis L and Ricinus communis aqueous extractsagainst vero cell line and evaluation of their antioxidant activi-tiesrdquo African Journal of Biotechnology vol 11 no 52 pp 11530ndash11534 2012

[30] R Ilavarasan M Mallika and S Venkataraman ldquoAnti-inflammatory and free radical scavenging activity of Ricinuscommunis root extractrdquo Journal of Ethnopharmacology vol 103no 3 pp 478ndash480 2006

[31] X-Y Zhou P Wu L Zhang et al ldquoEffects of lipoxin A4on

lipopolysaccharide induced proliferation and reactive oxygenspecies production in RAW2647 macrophages through mod-ulation of G-CSF secretionrdquo Inflammation Research vol 56 no8 pp 324ndash333 2007

[32] M Karlsson T Kurz U T Brunk S E Nilsson and C IFrennesson ldquoWhat does the commonly used DCF test foroxidative stress really showrdquo Biochemical Journal vol 428 no2 pp 183ndash190 2010

[33] A K Saini R Goyal V K Gauttam and A N Kalia ldquoEvalu-ation of anti-inflammatory potential of Ricinus communis Linnleaves extracts and its flavonoids content inWistar ratsrdquo Journalof Chemical and Pharmaceutical Research vol 2 no 5 pp 690ndash695 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


atherosclerosis aging immunosuppression ischemic heartdisease diabetes hair loss membrane lipid peroxidationand decreased membrane fluidity [3 6ndash8] Reactive oxygenspecies are also reported as carcinogenic and mutagenicagents [9] Signs of inflammation are swollen joints jointpain stiffness and loss of joint functions Nonsteroidalanti-inflammatory drug (NSAIDs) such as ibuprofen andnaproxen are anti-inflammatory drugs currently used fortreatment of inflammation These drugs are known to causesevere side effects in the body such as heart attacks and stroke[10]

Plant constituents are responsible for both free radi-cals scavenging and anti-inflammatory activity Secondarymetabolites are responsible for biological activities of plantsincluding terpenoids [11] phenolic compounds (flavonoidsphenolic acids quinones coumarins lignans stilbenes tan-nins) and nitrogen compounds (alkaloids amines andbetalains) and carotenoids [12] Iqbal et alrsquos [13] reports areavailable which suggest that phenolic compounds are thepotent antioxidant compounds from medicinal plants

Ricinus communis (Castor oil plant) belongs to the familyEuphorbiaceae Castor oil plant is originally from Africa andis now found in all tropical countries [14] Leaves barksseeds roots and oil of the plant have been traditionally usedfor many purposes throughout the world The leaf root andseed oil of the plant have also been used for treatment ofinflammation and liver disorders in India [15] In TunisiaR communis is used as a contraceptive herbal drug fortreatment of cold and tumors as a hypoglycemic and as alaxative R communis is well known for its biological activi-ties most important of which are hepatoprotective laxativeantidiabetic and antifertility activities [16] Literature searchrevealed that essential oils from R communis have beenreported to have potential antimicrobial anticarcinogenicantioxidant and antidiabetic activities [17ndash19]The aimof thisstudy was to evaluate the cytotoxic effects of R communisextracts against Human Caucasian skin fibroblast (BUD-8)cell line as well as its antioxidant activityusing 221015840-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) freeradicals and anti-inflammation activity on the Raw 2647macrophages cell line

2 Methods and Materials

21 Plant Collection and Processing Leaves were collectedin summer of 2011 from University of Limpopo (Turfloopcampus) South Africa Voucher specimens in the gardenHerbarium and tree labels verified the identity of the plantPlant was confirmed by Dr Brownyn Egan (Herbarium) andthe accession detail was UNIN 11352 The plant was selectedbased on the high antioxidant activity demonstrated inprevious in vitro study [20] Leaves were separated from twigsand dried at room temperature The dried plant materialswere milled to fine powder and stored at room temperaturein closed bottles in the dark until use for the extraction

22 Extraction Procedure Dried plant materials weresequentially extracted by mixing 10 g of plant material with

100mL of hexane dichloromethane acetone and methanolin glass bottle Bottles were vigorously shaken at a speed of300 rpm overnight Plant residues were allowed to settleand the supernatant was filtered and evaporated usinga rotary evaporator (BUCHi Labotec rotavapor modelR-205 Germany) and transferred into preweighed labeledglass beakers The process was repeated three times toexhaustively extract the plant material and the extracts foreach solvent were combinedThe solvent was removed undera stream of cold air at room temperature Plant extractswere reconstituted using acetone to a final concentration of10mgmL

23 Analysis of Extracts by TLC Ten microliters of10mgmL plant extracts were loaded on a thin layerchromatography plates Three separation systems of varyingpolarities were used to analyse plant extracts by thinlayer chromatography (TLC) (Fluka silica gel F

254plates)

benzene ethanol ammonium hydroxide (BEA) (36 4 04)chloroform ethyl acetate formic acid (CEF) (20 16 4)and ethyl acetate methanol water (EMW) (20 108 8)Chromatograms were examined under ultraviolet light (254and 365 nm) and sprayed with freshly prepared vanillin sprayreagent (01 g vanillin 28mL methanol and 1mL sulphuricacid) to visualise separated compounds The plates werecarefully heated at 110∘C for optimal colour development[21]

24 Antioxidant Activities

241 Qualitative 22-Diphenyl-1-picrylhydrazyl Assay on TLCThin layer chromatography plates were used to separate crudeextracts as described in Section 23 To detect antioxidantactivity chromatograms were sprayed with 02 (wv) 22-diphenyl-1-picrylhydrazyl (DPPH) (Sigma) in methanol asan indicator Yellow zones appearing on the chromatogramsindicate the presence of scavenging activity of free radicals bycompounds presence in plant extracts [22]

242 Quantitative Antioxidant Assay Using ABTS+ A stocksolution of 7mM of ABTS+ was prepared according to vanden Berg et al [23] by mixing 8mg of ABTS with 1mL ofwater ABTS free radicals were produced by reacting equalvolume of ABTS stock solution with 132mg in 10mL of(245mM) potassium sulphate The solution was preparedand incubated for 12ndash16 hours in the dark at room tempera-ture until the reaction was complete and stored at 4∘C untiluse Serial dilution method was used to prepare differentconcentrations of crude extracts (hexane dichloromethaneacetone and methanol) ABTS+ was diluted with waterfor equilibration of absorbance 070 (plusmn002) at 734 nm andappropriate blank was used without test samples Hundredmicroliters of ABTS+ was added to 100120583L of 250 125 063016 008 and 004mgmL concentrations of crude extractsThe absorbance reading was taken after 6 minutes following


BEA

CEF

EMW




(a)

Dichloromethane

BEA

CEF

EMW




(b)






(1)




50














0

20

40

60

80

100

120

004 008 016 031 063 125 250

Scav

engi

ng ac

tivity

()


HexaneDCM

AcetoneMethanol






0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol


Cel

l via

bilit

y (

)24hrs

(a)

0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol

Cel

l via

bilit

y (

)

48hrs


(b)









50for all crude



50















Methanol LPS PMA











2DCF fluorescence



4 Conclusion




Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BEA

CEF

EMW




(a)

Dichloromethane

BEA

CEF

EMW




(b)






(1)




50














0

20

40

60

80

100

120

004 008 016 031 063 125 250

Scav

engi

ng ac

tivity

()


HexaneDCM

AcetoneMethanol






0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol


Cel

l via

bilit

y (

)24hrs

(a)

0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol

Cel

l via

bilit

y (

)

48hrs


(b)









50for all crude



50















Methanol LPS PMA











2DCF fluorescence



4 Conclusion




Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















0

20

40

60

80

100

120

004 008 016 031 063 125 250

Scav

engi

ng ac

tivity

()


HexaneDCM

AcetoneMethanol






0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol


Cel

l via

bilit

y (

)24hrs

(a)

0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol

Cel

l via

bilit

y (

)

48hrs


(b)









50for all crude



50















Methanol LPS PMA











2DCF fluorescence



4 Conclusion




Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol


Cel

l via

bilit

y (

)24hrs

(a)

0

20

40

60

80

100

120

Control 100 200 300 400 500

HexaneDCM

AcetoneMethanol

Cel

l via

bilit

y (

)

48hrs


(b)









50for all crude



50















Methanol LPS PMA











2DCF fluorescence



4 Conclusion




Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Methanol LPS PMA











2DCF fluorescence



4 Conclusion




Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2DCF fluorescence



4 Conclusion




Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article assessment of cytotoxicity, antioxidant ...extracts against human caucasian skin...

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