ROS-Mediated Mitochondrial Pathway is Required for...

Research ArticleROS-Mediated Mitochondrial Pathway is Required forManilkara Zapota (L) P Royen Leaf Methanol Extract InducingApoptosis in the Modulation of Caspase Activation andEGFRNF-120581B Activities of HeLa Human Cervical Cancer Cells

Bee Ling Tan 1 Mohd Esa Norhaizan 123 and Lee Chin Chan4

1Department of Nutrition and Dietetics Faculty of Medicine and Health Sciences Universiti Putra Malaysia43400 Serdang Selangor Malaysia2Laboratory of Molecular Biomedicine Institute of Bioscience Universiti Putra Malaysia 43400 Serdang Selangor Malaysia3Research Centre of Excellent Nutrition and Non-Communicable Diseases (NNCD) Faculty of Medicine and Health SciencesUniversiti Putra Malaysia 43400 Serdang Selangor Malaysia4Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia43400 Serdang Selangor Malaysia

Correspondence should be addressed to Mohd Esa Norhaizan nhaizanupmedumy

Received 9 February 2018 Revised 23 April 2018 Accepted 15 May 2018 Published 6 June 2018

Academic Editor Luigi Milella

Copyright copy 2018 Bee Ling Tan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Manilkara zapota (L) P Royen (family Sapotaceae) is commonly called sapodilla or locally known as ciku The detailedmechanisms underlying Manilkara zapota leaf methanol extract against HeLa human cervical cancer cells have yet to beinvestigated Therefore our present study is designed to investigate the ability to induce apoptosis and the underlying mechanismsofManilkara zapota leaf methanol extract inducing cytotoxicity in HeLa cellsThe apoptotic cell death was assessed using AnnexinV-propidium iodide staining Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential activities weremeasured using dichlorodihydrofluorescein diacetate and MitoLite Orange respectively by NovoCyte Flow Cytometer Baxand Bcl-2 expression were evaluated using Enzyme-Linked Immunosorbent Assay Caspase-3 activity was determined using acolorimetric assay The associated biological interaction pathways were evaluated using quantitative real-time PCR Our datashowed that HeLa cells were relatively more sensitive toManilkara zapota leaf methanol extract than other cancer cell lines studiedOverall analyses revealed thatManilkara zapota leaf methanol extract can inhibit the viability of HeLa cells induce mitochondrialROS generation and inhibit nuclear factor-kappa B (NF-120581B) and epidermal growth factor receptor (EGFR) transcriptionalactivities Our results suggested thatManilkara zapota leaf methanol extract might represent a potential anticervical cancer agent

1 Introduction

Cervical cancer has become the fourth leading cancer inwomen contributing to approximately 530000 new casesin 2012 and represents 79 of all female cancers Nearly90 of the 270000 deaths from cervical cancer occurredin low- and middle-income countries in 2015 worldwide[1] Although locally advanced and early-stage diseasescan be cured by chemoradiotherapy and radical surgery[2] conventional therapy is not likely effective due to

undesirable effects thereby limiting their use in manypatients

Epidermal growth factor receptor (EGFR) has emergedas a crucial therapeutic target in more than 30 of solidtumors which is commonly associated with a poor prognosis[3] Apart from EGFR signaling nuclear factor-kappa B (NF-120581B) is a crucial transcription factor that has an ability toactivate the large array of inflammatory mediators and hasbeen recognized as a central player for cervical cancer [4]Notonly does NF-120581B activity stimulate tumor cell proliferation

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 6578648 19 pageshttpsdoiorg10115520186578648

2 Evidence-Based Complementary and Alternative Medicine

and inhibit apoptosis but it also facilitates metastasis [4]Given the pivotal role of NF-120581B in human cancer initiationdevelopment and metastasis NF-120581B pathway may serve as apromising therapeutic target

There have been extensive studies on natural compoundswhich showed a potent antiproliferative activity in con-junction with having good antioxidant activities [5 6]Many plants especially traditional medicinal plants havebeen widely investigated for their antioxidant activity inthe last few decades [7] Emerging evidence has demon-strated the role of natural antioxidant in the prevention ofchronic diseases including cancer and inflammation [8ndash10]In continuation of the efforts towards the discovery of bettertreatment strategies for cervical cancer plants have gainedremarkable interest as an effective anticancer agent In linewith this there is an unmet need to discover new anticanceragent with high efficacy and specificity but showing minimaladverse outcome

Manilkara zapota (L) P Royen (family Sapotaceae)commonly called sapodilla or locally known as ciku isan evergreen tree grown abundantly throughout India sub-continent including Bangladesh [11] though it is native toCentral America and Mexico Manilkara zapota leaf hasbeen traditionally used for the treatment of diarrhea coldand coughs [12] Nonetheless there is no pharmacologicalstudy on anticervical cancer properties of Manilkara zapotaleaf methanol extract in the literature Our earlier studydemonstrated that Manilkara zapota leaf water extract hascytotoxic activity towards human hepatocellular carcinoma(HepG2) and human colorectal adenocarcinoma (HT-29) celllines (unpublished data) Therefore this study was designedto investigate the ability to induce apoptosis and the under-lying mechanisms ofManilkara zapota leaf methanol extractinducing cytotoxicity in HeLa cells These molecular interac-tions underlying the apoptotic mediated signaling pathwayin cellular function may be involved in the modulation ofcervical cancer and deserve further elucidation

2 Materials And Methods

21 Chemicals and Reagents RPMI-1640 mediumMycoplex fetal bovine serum (FBS) penicillin andstreptomycin (100times) Dulbeccorsquos Modified Eagle Medium(DMEM) and trypsin-ethylenediaminetetraacetic acid(EDTA) (1times) were bought from Gibco (Grand IslandNY USA) Cycle TEST PLUS DNA Reagent Kit andAnnexin V-FITC Apoptosis Detection Kit I were procuredfrom BD Biosciences Pharmingen (Franklin Lakes NJUSA) Mitochondrial Membrane Potential Assay Kit(orange fluorescence) was bought from Abnova (Taipei CityTaiwan) Bax and Bcl-2 Human SimpleStep ELISAKits wereobtained from Abcam UK Caspase Colorimetric Assay Kitwas bought fromRampD Systems (MinneapolisMN USA) Allother reagents and chemicals used were of analytical gradeand obtained from Sigma-Aldrich (St Louis MO USA)

22 Plant Materials The plant (Manilkara zapota (L) PRoyen) was collected from Pahang Malaysia The plant

with voucher specimen number SK 317917 was deposited inBiodiversity Unit of Institute of Bioscience Universiti PutraMalaysia

23 Preparation of Plant Extract Initially leaf of Manilkarazapota was cut into small pieces and dried in an oven at40∘C for 3 days before being ground into powder formManilkara zapota leaf sample was extracted with methanolfollowing the method of Tan et al [13] About 5 g ofsamples was extracted with 40 mL of methanol in a shaker(Heidolph Inkubatorhaube Germany) at 40∘C for 2 h Theslurry was filtered using filter paper (Whatman no 1) Thefiltrate from the methanol extract was dried using rotaryevaporator (Buchi Rotavapor R-200 Switzerland) The yieldwas measured using electronic balance (Shimadzu KyotoJapan) and stored at -20∘C until further analyses The yieldof plant extract was calculated as follows

Percentage of plant yield ()

=Weight of plant extract (g)Weight of plant sample (g)

times 100(1)

24 Cell Culture The human colon carcinoma (HCT-116)human colorectal adenocarcinoma (HT-29) human cervicalcancer (HeLa) human hepatocellular carcinoma (HepG2)human gastric adenocarcinoma (HGT-1) human prostatecancer (PC-3) and mouse fibroblast (BALBc 3T3) celllines were obtained from American Type Culture Collection(ATCC Rockville MD USA) The HT-29 HCT-116 andHGT-1 cells were grown in DMEM supplemented with 100IUmL penicillin 100 120583gmL streptomycin and 10 (vv)FBSHeLaHepG2 PC-3 andBALBc 3T3 cells were culturedin RPMI-1640 medium supplemented with 100 IUmL peni-cillin 100 120583gmL streptomycin and 10 (vv) FBS All celllines were grown at 5CO2 atmosphere and 37∘Chumidifiedatmosphere incubator

25 Determination of Cell Viability using 3-(45-Dimethyl-thiazol-2-yl)-25-diphenyltetrazolium Bromide (MTT) AssayThe cell viability of HT-29 HCT-116 HeLa HGT-1 HepG2PC-3 and BALBc 3T3 upon treatment with Manilkarazapota leaf methanol extract was determined using MTTassay [13] HT-29 HCT-116 HeLa HGT-1 HepG2 PC-3 andBALBc 3T3 cells were seeded at a density of 5 times 104 cellswellin a 96-well plate After an overnight incubation the cellswere exposed to leaf methanol extract of Manilkara zapota(156-200 120583gmL) Untreated BALBc 3T3 and cancer celllines were included Following 24 48 and 72 h of treatment20 120583L of MTT (5 mgmL) was added to each well followedby 2-4 h incubation Lastly the media from each well werediscarded and 100 120583L of dimethyl sulfoxide (DMSO) wasadded to solubilize the purple-blue formazanThe absorbancewas read at 570 nm using an ELISA microplate reader(Tecan Switzerland) and 630 nm was used as a referencewavelength A graph of percentage of cell viability versusconcentration of Manilkara zapota leaf methanol extractwas plotted and the concentration of Manilkara zapota leafmethanol extract which inhibited 50 of cellular growth

Evidence-Based Complementary and Alternative Medicine 3

as compared to the control (50 inhibitory concentration(IC50)) was determined The cell viability was calculated asfollowsPercentage of cell viability ()

= OD570-630 treatmentOD570-630 control

times 100

OD = Optical density

(2)

26 Determination of Lactate Dehydrogenase Assay Cellcytotoxicity was evaluated using an in vitro Toxicology AssayKit by the release of lactate dehydrogenase (LDH) followingthe manufacturerrsquos protocol The HCT-116 HT-29 HeLaHGT-1 HepG2 PC-3 and BALBc 3T3 cell lines were seededat a density of 5 times 104 cellswell in a 96-well plate After24 h the cells were exposed to different concentrations ofManilkara zapota leaf methanol extract (156-200 120583gmL)for 24 48 and 72 h and subsequently the supernatant wascollected and used to measure the LDH activity UntreatedBALBc 3T3 and cancer cell lines were included The LDHmixtures were added to each well in a volume equal to twicethe volume of medium discarded The reaction was haltedafter addition of 110 (vv) of 1 N HCl into each well Theabsorbance was measured using ELISA microplate reader(Tecan Switzerland) at a wavelength of 490 nm

27 Determination of Cell Morphological Changes The HeLacells were seeded at a density of 1 times 106 cellsmL in a 6-well plate After an overnight incubation the HeLa cells wereexposed to 12 24 and 48 120583gmL of Manilkara zapota leafmethanol extract for 24 48 and 72 h The morphologicalchanges and the characteristic of necrosis or apoptosis of theuntreated HeLa cells and HeLa cells induced withManilkarazapota leaf methanol extract were viewed under an invertedlight microscope (Olympus Center Valley PA USA)

28Determination of Cell CycleArrest by FlowCytometer Thecell cycle arrest was measured using CycleTEST PLUS DNAReagent Kit following themanufacturerrsquos protocolTheHeLacells were seeded at a density of 1 times 106 cells in 25 cm2 tissueculture flask After an overnight incubation the cells weretreated with 12 24 and 48 120583gmL of Manilkara zapota leafmethanol extract for 72 h HeLa cells were then centrifugedat 30 times g for 5 min at room temperature followed by theaddition of buffer solution 250 120583L of solution A (trypsinbuffer) and 200 120583L of solution B (trypsin inhibitor and RNasebuffer) were subsequently added to the cells followed by10 min incubation at room temperature respectively Themixture was then mixed with cold solution C (200 120583L ofpropidium iodide (PI) stain solution) followed by incubationat 4∘C for 10 min The cells were filtered using a 40-120583m cellstrainer cap Data acquisition and analysis were measuredusing NovoCyte Flow Cytometer (ACEA Biosciences Inc)with NovoExpress software

29 Determination of Apoptosis by Annexin V-PropidiumIodide Staining The early and late apoptotic cells activity

were evaluated using Annexin V-FITC Apoptosis Detec-tion Kit I following the manufacturerrsquos instruction HeLacells were seeded at a density of 1 times 106 cells in 25cm2 tissue culture flask After an overnight incubationthe cells were exposed to 12 24 and 48 120583gmL ofManilkara zapota leaf methanol extract for 72 h Afterincubation for 72 h the cells were trypsinized and rinsedtwicewith phosphate-buffered saline-bovine serumalbumin-ethylenediaminetetraacetic acid (PBS-BSA-EDTA) and thecell pellet was resuspended in 100 120583L of 1 times binding buffer(01MHepesNaOH pH 74 and 14MNaCl2 25mMCaCl2)An aliquot of 10 120583L of PI and 5 120583L of Annexin V-fluoresceinisothiocyanate (FITC) was added to each sample prior toincubation for 10 min in the dark Lastly 400 120583L of 1 timesbinding buffer was mixed to the cells and the fluorescencewas determined using NovoCyte Flow Cytometer (ACEABiosciences Inc) with NovoExpress software

210 Determination of Bax and Bcl-2 Activities in ManilkaraZapota Leaf Methanol Extract The Bax and Bcl-2 activitieswere evaluated using Bax andBcl-2 human SimpleStep ELISAKits following themanufacturerrsquos protocol BrieflyHeLa cellswere seeded in 25 cm2 tissue culture flask at a density of 1times 105cells After an overnight incubation the cells were exposedto 12 24 and 48 120583gmL of Manilkara zapota leaf methanolextract for 72 h The cells were collected and centrifuged at500times g at 4∘C for 5min to discard themediumThe cells wererinsed twice with phosphate-buffered saline (PBS) and cold1timesCell Extraction Buffer PTR and subsequently incubated onice for 20minThe cell lysates were then centrifuged at 18000times g and 4∘C for 20 min and the supernatants were collectedBradford protein assay kit was used to quantify the proteinconcentrationAn aliquot of the samplewas diluted to desiredconcentration using 1times Cell Extraction Buffer PTR About50 120583L of standard or sample was then mixed with 50 120583L ofantibody cocktail in each well of 96-well plate The plate wassealed followed by incubation at room temperature for 1 h ona plate shaker set to 400 times g Each well was rinsed with 3times 350120583L 1times wash buffer PT An aliquot of 100 120583L of TMB substratewas added to each well followed by 10 min incubation in thedark on a plate shaker set to 400times g Next 100120583Lof Stop Solu-tion wasmixed into each wellThe plate was shaken on a plateshaker for 1 min and measured at the wavelength of 450 nm

211 Determination of Caspase-3 Assay The caspase-3 activ-ity wasmeasured spectrophotometrically using a commercialcolorimetric assay kit followed by spectrophotometric detec-tion of the chromophore pnitroanilide (pNA) after cleavageof the specific substrates DEVD-pNA (for caspase-3) BrieflyHeLa cells were seeded at a density of 1 times 105 cells in a 6-wellplate After an overnight incubation the cells were exposedto 12 24 and 48 120583gmL of Manilkara zapota leaf methanolextract for 72 h The cells were centrifuged at 250 times g for 10min to discard the medium The cell pellets were then lysedin 25 120583L of cold lysis buffer followed by 10 min incubationon ice The cell lysates were then centrifuged at 10000 timesg and 4∘C for 1 min and the supernatants were collectedBradford protein assay kit was used to quantify the protein


concentration An aliquot of 50 120583L of 2times Reaction Buffer 3(prior to using the 2times Reaction Buffer 3 10 120583L of DTT wasmixed with 1 mL 2times Reaction Buffer 3) was mixed with 50120583L of cell lysate containing 200 120583g of total protein followedby 5 120583L of caspase-3 colorimetric substrate (DEVD-pNa)Subsequently the reaction mixture was kept at 37∘C for 2 hbefore being analyzed using ELISAmicroplate reader (TecanSwitzerland) at a wavelength of 405 nm

212 Determination of Intracellular Reactive Oxygen Species inManilkara Zapota Leaf Methanol Extract Induces OxidativeStress The intracellular reactive oxygen species (ROS) inHeLa cells treated with Manilkara zapota leaf methanolextract wasmeasured using dichlorodihydrofluorescein diac-etate (DCFH-DA) Initially HeLa cells were seeded at adensity of 1 times 105 cellswell in a 6-well plate overnight andpreincubated with 10 120583M DCFH-DA in complete media for1 h DCFH-DA was discarded and washed twice with PBSfollowed by treatment with Manilkara zapota leaf methanolextract (12 24 and 48 120583gmL) for 72 h Following 72 h ofincubation all adherent and floating cells were collectedThe sample was analyzed using NovoCyte Flow Cytometer(ACEA Biosciences Inc) with NovoExpress software

213 Determination of Mitochondrial Membrane PotentialThe Mitochondrial Membrane Potential Assay Kit (orangefluorescence) (Abnova Taipei City Taiwan) was used tomeasure the alteration of mitochondrial membrane potential(MMP) Initially HeLa cells were seeded at a density of 1times 105 cellswell in a 25 cm2 tissue culture flask After anovernight incubation the cells were exposed to 12 24 and48 120583gmL of Manilkara zapota leaf methanol extract for 72h After treatment the cells were trypsinized rinsed twice inPBS and the cells were suspended in 1 mL of Assay BufferAfter adding 2 120583L of 500times MitoLite Orange the cells wereincubated at 37∘C and 5 CO2 incubator for 30 min Thecells were centrifuged at 500 times g for 4 min Lastly the cellswere resuspended in 1 mL of Assay Buffer The fluorescenceintensity was measured using NovoCyte Flow Cytometer(ACEA Biosciences Inc) with NovoExpress software

214 Determination of Catalase Activity Initially HeLa cellswere seeded at a density of 1 times 105 cells for 24 hThe cells werethen treated with 12 24 and 48 120583gmL of Manilkara zapotaleaf methanol extract for 72 h HeLa cells were centrifuged at250 times g for 10 min to discard the supernatant The cell pelletswere subsequently lysed in 100 120583L of cold lysis buffer prior tobeing incubated for 10 min on ice Following centrifugationat 10000 times g and 4∘C for 1 min the supernatants werecollected and kept at -80∘C for catalase assay Catalase levelwas determined using the method described by Aebi [14]A 19-mL aliquot of the phosphate buffer (005 M pH 70)was mixed with 01 mL of supernatant and 1 mL of hydrogenperoxide (0019 M) The absorbance was measured at awavelength of 240 nm using UV-visible spectrophotometer(PharmaspecUV-1700 Shimadzu Kyoto Japan)The catalaseactivity was expressed as nmol H2O2 consumed minminus1 mgminus1protein

215 Total RNA Extraction and cDNA Synthesis Totalribonucleic acid (RNA) was isolated using TRI Reagentfollowing themanufacturerrsquos instructionTheHeLa cells wereseeded at a density of 1 times 105 cells in a 25 cm2 culture flaskfor 24 h After being treated with 12 24 and 48 120583gmL ofManilkara zapota leafmethanol extract for 72 h the cells werehomogenized and the cell lysates were aliquot in falcon tubesAn aliquot of 1mLTRIReagentwas added in falcon tubes andresuspended About 100 120583L of 1-bromo-3-chloropropane permL of TRI Reagent used was added and vortexed vigorouslyfor 15 s prior to incubation for 2ndash15min at room temperatureAfter centrifugation for 15000 times g and 2-8∘C for 15 minthe mixture was divided into a lower red organic layer aninterphase and a colorless upper aqueous layer containingRNA The aqueous layer was precipitated after the additionof 500 120583L of isopropanol The sample was kept for 5-10 minat room temperature before being centrifuged at 11500 times gand 2-8∘C for 10 min The supernatant was discarded andthe RNA pellet was rinsed with 1 mL of 75 (vv) ethanolfollowed by centrifugation at 5500 times g and 2-8∘C for 5 minAn aliquot of 50 120583L of RNase-free water wasmixed with RNApellet and resuspended before being stored at -80∘C About2 120583g of total RNA per 20 120583L was reverse-transcribed usingHigh Capacity RNA-to-cDNA Kit following the manufac-turerrsquos instructions The reverse transcription reaction wasconducted using an AuthorizedThermal Cycler

216 Quantitative Real-Time Polymerase Chain ReactionAnalysis Quantitative real-time PCR was performed usingSYBR SelectMasterMix (CFX) Table 1 shows the nucleotideprimer sequences originating from human cell lines Thespecific primers were validated for amplification efficiencyover a concentration range and consistency with the ampli-fication efficiency of housekeeping genes and amplificationspecificity The mRNA levels of cytochrome c epidermalgrowth factor receptor (EGFR) and nuclear factor-kappa B(NF-120581B) were assayed using SYBR Select Master Mix CFXin a final volume of 20 120583L following the manufacturerrsquosinstructions Briefly the primers kit contents (RNase-freewater and SYBR Select Master Mix (CFX)) and cDNA tem-plate were thawed on iceThe qPCR reaction was determinedbased on the following conditions (1) 50∘C for 120 s (1cycle) for uracil-DNA glycosylase (UDG) activation (2) 95∘Cfor 120 s (1 cycle) for DNA polymerase activation (3) 95∘Cfor 2 s (40 cycles) for denaturation and (4) 60∘C for 30s (40 cycles) for annealingextension All the controls andsamples were evaluated in triplicate using the BioRAD-iQ5 Multicolor Real-Time PCR Detection System (HerculesCA USA) and CFX Manager software (version 16 Bio-Rad Hercules CA USA) was used for data analysis Thehousekeeping genes (18S rRNA glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and beta-actin (ACTB)) were usedfor normalization

217 Determination of Total Phenolic Content Total phenoliccontent (TPC) in plant extract was measured by using Folin-Ciocalteursquos reagent following a modified method by Medaet al [15] An aliquot of 03 mL of plant extract was mixed


Table 1 Nucleotide sequence of PCR primers for amplification and sequence-specific detection of cDNA (obtained fromGenBank database)

Primer name [accession number] Oligonucleotides (51015840-31015840) sequence

Cytochrome c [JF9192241] F ATCACCTTGAAACCGACCTGR CTCCCTGAGGATAACGCAAA

EGFR [NM 0052283] F CAGCGCTACCTTGTCATTCAR TGCACTCAGAGAGCTCAGGA

NF-120581B [M58603] F TGGAAGCACGAATGACAGAGR TGAGGTCCATCTCCTTGGTC

ACTBa [NM 0011013] F AGAGCTACGAGCTGCCTGACR AGCACTGTGTTGGCGTACAG

GAPDHa [NM 0020464] F GGATTTGGTCGTATTGGGCR TGGAAGATGGTGATGGGATT

18S rRNAa [HQ3870081] F GTAACCCGTTGAACCCCATTR CCATCCAATCGGTAGTAGCG

ACTB beta-actin EGFR epidermal growth factor receptor GAPDH glyceraldehyde-3-phosphate dehydrogenase NF-120581B nuclear factor-kappa BaHousekeeping gene

with 12 mL of sodium carbonate (75 (wv)) and 15 mLof Folin-Ciocalteursquos reagent (diluted 10 times) The mixturewas vortexed prior to being incubated at room temperaturefor 30 min The absorbance of the sample was read at 765nmusing aUVndashvisible spectrophotometer (PharmaspecUV-1700 Shimadzu Kyoto Japan) The TPC value of the samplewas expressed in milligram gallic acid equivalents per gramof extract (mg GAEg extract)

218 Determination of Total Flavonoid Content Totalflavonoid content (TFC) in plant extract was measuredfollowing a modified method by Shanmugapriya et al [16]An aliquot of 05 mL of plant extract solution was addedto 01 mL of 10 aluminium chloride hexahydrate and 15mL of 95 ethanol The mixture was then added to 28mL distilled water and 01 mL of potassium acetate (1 M)The absorbance of the sample was read at 415 nm after 40min incubation at room temperature An equal volume (01mL) of distilled water was substituted with 10 aluminiumchloride hexahydrate as the blank The total flavonoidcontent was expressed in milligram quercetin equivalentsper 100 gram of extract (mg QE100 g)

219 Beta-Carotene Bleaching Test The 120573-carotene bleachingassay was performed as previously described by Tan et al[13] An aliquot of 5 mg of 120573-carotene was added to 10 mLof chloroform and 3 mL of this mixture was transferred to100 mL round-bottom flask The chloroform was evaporatedat 40∘C by vacuum evaporation After evaporation about 40mg of linoleic acid and 400 mg of Tween 40 emulsifier weremixed with 100 mL of distilled water followed by vigorousshaking A 48-mL aliquot of this emulsion was mixedwith extract or 200 120583L of methanol (control) The standardantioxidant used was butylated hydroxytoluene (BHT) Afterthe addition of this emulsion into a series of test tubes thezero time absorbance was read at a wavelength of 470 nmusing UV-visible spectrophotometer (Pharmaspec UV-1700Shimadzu Kyoto Japan) The subsequent absorbance wasread over 2 h periods at every 20 min followed by incubationof the test tubes at 50∘C The blank samples were used forbackground subtraction The capacity of the plant extractto protect against 120573-carotene oxidation was calculated asfollows

((At-0 Sample minus At-0Blank) ndash (At-120min Sample minus At-120minBlank))((At-0Control) minus (At-120minControl))

= C

Beta-carotene retention () = 100 minus (C) times 100

A = absorbance at a particular time C = carotene depletion factor

(3)

220 Determination of 1 1-Diphenyl-2-picryl-hydrazyl(DPPH) Radical Scavenging Capacity The 11-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging capacity wasevaluated by UV-visible spectrophotometer (PharmaspecUV-1700 Shimadzu Kyoto Japan) [17] A 15-mL aliquot

of DPPH (01 nM) in methanol was mixed with 05 mLsample The solution was vortexed for 15 s prior to beingincubated at room temperature for 1 hThe absorbance of thesample was read at a wavelength of 517 nm Control (withoutsample) and standard (ascorbic acid) were prepared using


the same methodology DPPH assay is expressed as effectiveconcentration (EC50) the concentration which is needed toscavenge 50 of the DPPH free radicals

221 Determination of Polyphenols using Ultra PerformanceLiquid Chromatography (UPLC) Polyphenols quantificationin plant extract was carried out using Agilent Technologies1290 Infinity model G4220A equipped with a diode arraydetector setup wavelength of 280 nm and 320 nm Chromato-graphic separation was analyzed using a LiChroCART 250-4 6C18 column (5120583m 250mmtimes 46mm) Solvent (A)water-acetic acid (946 vv pH 227) and solvent (B) acetonitrilewere used as the mobile phase These gradient elution condi-tion and solvent composition have been described earlier byTan et al [18] The solvent gradients were as follows 0-15 Bfor 40 min 15-45 B for 40 min and 45-100 B for 10 minwith a flow rate of 05 mLmin An aliquot of 20 120583L samplewas injectedThemobile phase and sample were filtered usinga Millipore filter of 022 120583m The polyphenolic compoundswere quantified by comparing their retention times with thecalibration curves of their respective standards (caffeic acidsyringic acid vanillic acid ferulic acid gallic acid and p-coumaric acid)

222 Qualitative Analysis of Phytochemicals The leafmethanol extract of Manilkara zapota was analyzed for thepresence of flavonoids steroids saponins phlobatanninsand triterpenoids following the methods as previouslyreported by Harborne [19] and Evans [20]

2221 Qualitative Analysis of Steroids Steroids inManilkarazapota leaf methanol extract were determined using Salk-woskirsquos test About 05 g of the plant extract was mixed with2 mL of chloroform The sulphuric acid was added to themixture to form a layer A reddish brown color formed at theinterface which shows the presence of steroids

2222 Qualitative Analysis of Triterpenoids Triterpenoids inManilkara zapota leaf methanol extract were evaluated usingHishornrsquos test About 05 g of the plant extract was added to 2mL of chloroformThemixture was then mixed with 2 mL oftrichloroacetic acid (TCA) followed by incubation for 10minThe changes from yellow to red color indicate the presence oftriterpenoids

2223 Qualitative Analysis of Flavonoids Flavonoids weredetermined using ferric chloride test About 05 g of theManilkara zapota leafmethanol extract was boiled in distilledwater prior to being filtered Two or three drops of 10 ferricchloride were subsequently mixed with 2 mL of the filtrate Agreen-blue or violet color shows the presence of flavonoids

2224 Qualitative Analysis of Saponins Saponins inManilkara zapota leaf methanol extract were evaluated usingfrothing test About 1 g of the plant extract was mixed with 3mL of distilled water and vortexed vigorously for 5 min Thepresence of frothing indicates the presence of saponins

2225 Qualitative Analysis of Phlobatannins The presenceof a red precipitate after the plant extract boiled with 1hydrochloric acid indicates the presence of phlobatannins

223 Statistical Analysis The data are presented as the meanplusmn standard deviation (SD) using one-way analysis of variance(ANOVA) The differences with P lt 005 were consideredsignificant The statistical analyses were carried out using theStatistical Package for Social Science (SPSS) version 190

3 Results and Discussion

31 The Yield of Manilkara Zapota Leaf Methanol ExtractExtraction yield does depend on the extraction methodbut also on the extraction solvent Polar solvents are com-monly used for recovering polyphenols from plant matricesMethanol has been reported to bemore efficient in the extrac-tion of low molecular weight polyphenols [21] It can be seenthat the extraction yield of puremethanol (3106 plusmn 154) wassignificantly higher than that of 70 ethanol (837 plusmn 040)and water (876 plusmn 146) (P lt 005) (unpublished data) Thisresult indicates that compounds other than phenolic mayhave been extracted and thus contribute to the high yield

32 Manilkara Zapota Leaf Methanol Extract DecreasesViability of HeLa Cells To determine the antiproliferativeeffect of Manilkara zapota leaf methanol extract on cancercells human colon carcinoma (HCT-116) human colorectaladenocarcinoma (HT-29) human cervical cancer (HeLa)human gastric adenocarcinoma (HGT-1) human hepato-cellular carcinoma (HepG2) human prostate cancer (PC-3) and mouse fibroblast (BALBc 3T3) cell lines wereexposed to different concentrations ofManilkara zapota leafmethanol extract (156-200 120583gmL) for 24 48 and 72 hand the effects on cell viability were evaluated using 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay We found thatManilkara zapota leaf methanolextract was cytotoxic to all cancer cells studied after 72 hincubation (Table 2) According to published guidelines anyextract that possesses potentially cytotoxic activity shouldhave an IC50 less than 100 120583gmL [22] As shown in Table 2Manilkara zapota leaf methanol extract inhibited the growthof HT-29 cells after 24 48 and 72 h with IC50value 9327plusmn 1719 8929 plusmn 601 and 6912 plusmn 810 120583gmL respectivelyConsistent with the cytotoxic effect observed in HT-29 cellsManilkara zapota leaf methanol extract also decreases theviability of HCT-116 cells in a time-dependent manner after24 h (9014 plusmn 1423 120583gmL) 48 h (8733 plusmn 929 120583gmL) and 72h (8317 plusmn 992 120583gmL) A similar trend was also observed inHGT-1 and HepG2 cells We found that after treatment withManilkara zapota leaf methanol extract for 72 h both HGT-1 (4944 plusmn 1062 120583gmL) and HepG2 (7302 plusmn 933 120583gmL)cells were inhibited HeLa cells were relatively more sensitiveto Manilkara zapota leaf methanol extract than other cancercell lines studied It suppressed the viability of HeLa cells ina time-dependent manner with IC50 values 8929 plusmn 18205923 plusmn 1033 and 2387 plusmn 502 120583gmL for 24 48 and 72 hrespectively Figure 1(a) shows the percentage of viable HeLa


Table2Treatm

ento

fManilkarazapota

leafmethano

lextract(156-200120583g

mL)

onselected

cancer

celllin

esfor2

448and

72hevaluatedby

MTT

andLD

Hassays

Cancer

celllin

esMTT

(120583gmL)

LDH(120583gmL)

24h

48h

72h

24h

48h

72h

HT-29

9327plusmn1719

a8929plusmn601

a6912plusmn810

b9033plusmn1579a

8599plusmn487

a7622plusmn539

b

HCT

-116

9014plusmn1423a

8733plusmn92

9a8317plusmn99

2a9322plusmn90

3a9012plusmn97

7a8811plusmn1169a

HeLa

8929plusmn1820a

5923plusmn1033

a2387plusmn502

b8733plusmn1498a

8044plusmn1165a

2576plusmn893

b

HGT-1

8011plusmn1019

a7204plusmn523

a4944plusmn1062b

6520plusmn1427a

6211plusmn629

a5989plusmn1027a

HepG2

9729plusmn326

a8395plusmn92

0ab

7302plusmn93

3b8945plusmn1682a

8303plusmn535

a7704plusmn99

3a

HCT

-116h

uman

coloncarcinom

aHeLa

human

cervical

cancerH

epG2

human

hepatocellu

larcarcinom

aHGT-1hu

man

gastr

icadenocarcino

ma

HT-29h

uman

colorectal

adenocarcino

ma

LDHlactate

dehydrogenaseandMTT

3-(45-dimethylth

iazol-2

-yl)-25-diph

enyltetrazolium

brom

ide

Values

arerepo

rted

asmeanplusmnSD

(n=3)V

alue

with

different

superscriptletterinthesamerowfortheirrespectiv

eassayindicatessignificantd

ifference

byTu

keyrsquos

test(Plt005)In

MTT

assaytre

atmentw

ithManilkarazapota

leafmethano

lextractfor7

2h(691

2plusmn810120583gmL)

significantly

inhibitedthep

roliferationof

HT-29

cells

comparedto

24h(9327plusmn1719120583gmL)

(Plt005)whereasinLD

Hassaytherew

asa

significanteffectof

thecytotoxicactiv

ities

ofManilkarazapota

leafmethano

lextractin

HT-29

cells

incubatedfor7

2h(7622plusmn539120583gmL)

comparedto

thoseincubatedfor2

4h(9033plusmn1579120583gmL)

or48

h(8599plusmn487120583gmL)

(Plt005)


cells after 72 h exposure to Manilkara zapota leaf methanolextract Conversely we observed that Manilkara zapota leafmethanol extract promotes proliferation of PC-3 cells after24 48 and 72 h incubation (Figure 1(b)) Thus we believethat PC-3 cells were relatively more resistant to this extractcompared to other cancer cell lines However the molecularmechanisms underlying PC-3 cells in this extract warrantsfurther elucidation

To verify the cytotoxicity activity ofManilkara zapota leafmethanol extract the proliferation of all cancer cells studiedwas evaluated using lactate dehydrogenase (LDH) assay Cellstreated with different concentrations of Manilkara zapotaleaf methanol extract (156-200 120583gmL) were harvested andsubjected to LDH analysis Consistent with MTT resultsLDH analyses demonstrated that both cells viabilities ofHCT-116 and HT-29 were reduced after treatment withManilkara zapota leaf methanol extract (Table 2) Comparedto other cancer cell lines studied HeLa cells is the mostsensitive towards Manilkara zapota leaf methanol extractwith an IC50 value 2576 plusmn 893 120583gmL after 72 h incubationConversely HGT-1 and HepG2 cells were less sensitive com-pared to HeLa cells (Table 2) Consistent with the cytotoxiceffect observed in MTT assay our LDH analysis furtherdemonstrated that Manilkara zapota leaf methanol extractpromotes proliferation of PC-3 cells after 24 48 and 72 h(Figure 1(c)) Interestingly no cytotoxicity was observed inManilkara zapota leaf methanol extract in BALBc 3T3 celllines as evaluated using both MTT and LDH assays (Figures1(d) and 1(e)) Taken together our data suggest thatManilkarazapota leaf methanol extract can induce cytotoxicity indifferent cancer cell lines in which HeLa cells are being themost sensitive compared to other cancer cells studied ThusHeLa cells were selected for further analyses Given the widecytotoxicity range ofManilkara zapota leaf methanol extractagainst HeLa cells as evaluated using MTT and LDH assaysonly these three concentrations (12 24 and 48 120583gmL) wereselected for further analyses

33 Manilkara Zapota Leaf Methanol Extract Induces Mor-phological Changes of HeLa Cells To explore the morpho-logical changes of HeLa cells treated with Manilkara zapotaleaf methanol extract HeLa cells were exposed to differentconcentrations of the extract (12 24 and 48 120583gmL) Asdepicted in Figure 2 increasing concentration of Manilkarazapota leaf methanol extract from 12 to 48 120583gmL for 24 48and 72 h incubation led to cell morphological changes anddecrease in the number of cells (Figure 2) The proliferationof cells treated with 48 120583gmL of Manilkara zapota leafmethanol extract for 24 h and 48 h was inhibited and thisphenomenon became obvious at 72 h (Figure 2) The markeddetachment was observed in HeLa cells exposed to 12 120583gmLand in the latter (24 and 48 120583gmL) from 24 h to 72 h(Figure 3) Additionally we also observed cell roundingaccompanied with a typical apoptotic morphology includingchromatin condensation (CC) membrane blebbing (MB)apoptotic bodies (AB) nuclear fragmentation (NF) andnuclear compaction (NC) (Figure 3) Based on the findingsleaf methanol extract of Manilkara zapota induced cells

inhibition and showed the obvious typical characteristic ofapoptotic cells after 72 h incubationTherefore an incubationtime of 72 h was selected for further analyses

34 Manilkara Zapota Leaf Methanol Extract Induces CellCycle Arrest in HeLa Cells To examine if the cytotoxicactivity of Manilkara zapota leaf methanol extract was dueto the cell cycle arrest and induction of apoptosis HeLacells were exposed to different concentrations of Manilkarazapota leaf methanol extract (12 24 and 48 120583gmL) for 72h the cell apoptosis was evaluated by detecting the sub-G0 population upon propidium iodide (PI) staining andanalyzed by flow cytometry As illustrated in Figure 4(e)exponentially growing of untreated HeLa cells containeda low level (075) of apoptotic cells which is significantdifference between the untreated cells and those from thegroups treated with 12 120583gmL 24 120583gmL or 48 120583gmL ofManilkara zapota leaf methanol extract (P lt 005) Thisfinding indicates thatManilkara zapota leaf methanol extractinduces population sub-G0 phase following treatment withManilkara zapota leafmethanol extract (Figure 4(e)) indicateDNA degradation due to the activation of endogenousnucleases during apoptosis [23] Treatment with Manilkarazapota leaf methanol extract for 72 h significantly increasedthe percentage of cells at G0G1 phase as compared to theuntreated cells (P lt 005) with a concomitant decrease ofthe S phase at 72 h (Figure 4(e)) This result implies thatManilkara zapota leaf methanol extract regulates severalbiological processes associated with cell survival and deathOur findings presented in this study demonstrated thatManilkara zapota leafmethanol extract destroysHeLa cells individing state Overall both cell viability and flow cytometricassays suggest that Manilkara zapota leaf methanol extractcan indeed result in cytotoxicity

35 Manilkara Zapota Leaf Methanol Extract Induces Apopto-sis in HeLa Cells To further confirm Manilkara zapota leafmethanol extract induces apoptosis in HeLa cells AnnexinV-FITCPI double-staining followed by flow cytometry wasconducted in HeLa cells upon exposure toManilkara zapotaleaf methanol extract (12 24 and 48 120583gmL) for 72 h Weobserved that treatment with 24 and 48 120583gmL ofManilkarazapota leaf methanol extract led to a significant increasein the percentage of early apoptotic cells compared to thecontrol (P lt 005) (Figure 5(e)) The late apoptotic cellsin HeLa cells treated with 48 120583gmL Manilkara zapotaleaf methanol extract for 72 h were significantly increasedcompared to the control (P lt 005) Overall treatment with24 and 48 120583gmL Manilkara zapota leaf methanol extractfor 72 h significantly increased the total apoptotic HeLa cellscompared to the control (P lt 005) with a maximum effectnoted at a concentration of 48120583gmLOur results suggest thatManilkara zapota leaf methanol extract induces translocationof phosphatidylserine from inner to the outer leaflet of thecell membrane which indicates a hallmark of apoptosisImportantly we found that the percentage of total apoptoticcells was more prominent than necrotic cells (lt1) Thisfinding implied that Manilkara zapota leaf methanol extract


0 40 60 8020 160

180

100

120

200

140

Concentration (gmL)

010203040506070

Perc

enta

ge o

fvi

able

cells

()

(a)

aab ab ab ab b

c c

ab a ab abcbc

c

d d

aa a a a a

b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(b)

a a aa a

ab

bcc

aab abc

abc bc cdd

e

a a a ab b

b

c

24 h48 h72 h

25 50 100

200

625

125

156

3

312

5

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(c)

a a aa a a a a

a a a a a a a aa ab

ab ab ab ab b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(d)

24 h48 h72 h

a a a a aa a aa a a a a

a a aa ab ab ab ab ab b b

25 50 200

100

125

625

312

5

156

3Concentration (gmL)

020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(e)

Figure 1 Treatment ofManilkara zapota leaf methanol extract on cancer cells (a) Treatment ofManilkara zapota leaf methanol extract onHeLa cellsThe cell viability was evaluated by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay after 72 h exposurewith Manilkara zapota leaf methanol extract (b) Manilkara zapota leaf methanol extract increases proliferation of human prostate cancer(PC-3) cells after 24 48 and 72 h using MTT assay (c)Manilkara zapota leaf methanol extract promotes proliferation of PC-3 cells after 2448 and 72 h evaluated by lactate dehydrogenase (LDH) assay (d) Treatment of Manilkara zapota leaf methanol extract in mouse fibroblast(BALBc 3T3) cell lines evaluated using MTT assay (e) Cell viability of BALBc 3T3 cell lines after treatment with Manilkara zapota leafmethanol extract was evaluated using LDH assay Values are reported as mean plusmn SD (n = 3) Value with different superscript letter indicatessignificant difference between groups by Tukeyrsquos test (P lt 005)

might be used as a therapeutic agent for human cervicalcancer Taken together our data demonstrate thatManilkarazapota leaf methanol extract potentiates the apoptotic effectson HeLa cells rather than necrosis

36 Manilkara Zapota Leaf Methanol Extract Modulates Bcl-2Family in HeLa Cells To examine whether apoptosis induc-tion of Manilkara zapota leaf methanol extract in HeLa cellsinvolved the proapoptotic protein (Bax) and antiapoptoticprotein expression (Bcl-2) the Bax and Bcl-2 protein expres-sion in HeLa cells following treatment with 12 24 and 48120583gmLManilkara zapota leaf methanol extract was evaluated(Figures 6(a) and 6(b)) Extensive research has shown thatmany cellular organelles such as endoplasmic reticulummitochondria lysosomes and Golgi apparatus play a criticalrole in apoptotic cell death [24 25] The mitochondria have aparticularly prominent role in apoptosis andBcl-2 family pro-teins are central players in mitochondria-mediated cell death

and survival [26] Phosphorylation of Bcl-2 may be requiredto trigger its antiapoptotic functions [27] In the presentstudy the cells treated with 12 120583gmL ofManilkara zapota leafmethanol extract significantly increased the proapoptotic roleof Bax compared to the untreated cells (P lt 005) In additionour data also revealed that 48 120583gmL Manilkara zapotaleaf methanol extract induced the phosphorylation of Bcl-2suggesting that the Bcl-2 phosphorylation (Figure 6(b)) andBax activation (Figure 6(a))may correlate with apoptotic cellsdeath Taken together this finding indicates a crucial role ofthe Bcl-2 protein inManilkara zapota leaf methanol extract-induced apoptotic cell death

37 Manilkara Zapota Leaf Methanol Extract ActivatesCaspase-Dependent Apoptotic Pathway To verify whetherthe growth suppressive activity could be dependent on thestimulation of caspase-3 activity which plays a crucial role inthe regulation of apoptotic responses [28] the intracellular


Control 12 gmL 24 gmL 48 gmL72

hou

rs48

hou

rs24

hou

rs

50 m

50 m

50 m 50 m 50 m

50 m

50 m 50 m

50 m 50 m

50 m 50 m

Figure 2Manilkara zapota leaf methanol extract induces morphological changes and decreases the proliferation of human cervical cancer(HeLa) cells HeLa cells were incubated with 12 24 and 48 120583gmL of Manilkara zapota leaf methanol extract for 24 48 and 72 h and thenobserved under an inverted light microscope (Magnification 200times)

levels of caspase-3 in HeLa cells after exposure to Manilkarazapota leaf methanol extract (12 24 and 48 120583gmL) wereevaluated As shown in Figure 6(c) treatment withManilkarazapota leaf methanol extract showed apparently upregulationin caspase-3 activity compared to the control (P lt 005)Indeed quantification of caspase-3 enzymatic activity con-firmed the caspase activation by leaf methanol extract ofManilkara zapota Collectively these findings indicated thatManilkara zapota leaf methanol extract induced apoptosisinvolved caspase-dependent pathway in HeLa cell

38Manilkara Zapota LeafMethanol Extract Triggers Apopto-sis via ROS-Mediated Mitochondrial Pathway To investigatewhether the inhibitory effects of Manilkara zapota leafmethanol extract in HeLa cells involved oxidative stress weevaluated using reactive oxygen species (ROS) sensitive dyedichlorodihydrofluorescein diacetate (DCFH-DA) Our datapresented in this study showed that after exposure to 12 24and 48 120583gmL of Manilkara zapota leaf methanol extractsignificantly increased ROS generation as compared to theuntreated cells (control) (P lt 005) (Figure 7) This findingindicates that induction of intracellular ROS in HeLa cellswhich might be responsible for induction of apoptosis isdue to the oxidative stress induced by Manilkara zapota leafmethanol extract

Loss of mitochondrial membrane potential is an earlyevent during apoptosis Because excessive ROS accumu-lation may contribute oxidative stress and mitochondrial

dysfunction we evaluated mitochondrial function usingMitoLite Orange an indicator of mitochondrial membranepotential by flow cytometry analysis Our data revealedthat mitochondrial membrane potential in cells treated withManilkara zapota leaf methanol extract was significantlydecreased compared with the control (P lt 005) (Figure 8)These data indicate that Manilkara zapota leaf methanolextract induces depolarization andmitochondrial membranepotential collapse in cells leading to activation of apoptosisAs we know chemotherapy agents increase oxidative stressand result in ROS accumulation [29] The generation ofROS in the mitochondria could suppress the mitochondrialrespiration chain which causes mitochondrial membranerupture and apoptotic cell death [30] Collectively the datapresented in this study suggest that Manilkara zapota leafmethanol extract may modulate apoptosis through the ROS-mediated mitochondrial pathway

39 Manilkara Zapota Leaf Methanol Extract PromotesCatalase Activity To test whether the apoptotic effects ofManilkara zapota leaf methanol extract in HeLa cells maybe associated with the antioxidant enzyme we evaluatedthe catalase activity The decrease of catalase levels in theuntreated cells (control) (694 plusmn 001 nmol H2O2 consumedminminus1 mgminus1 protein) demonstrated that the defense mecha-nism may have been overwhelmed to ameliorate the amountof hydrogen peroxide ions generated on the surface of thecells The observed effect may also be due to the impairment


50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs

Figure 3 Close-up view of morphological changes in HeLa cells after treatment with Manilkara zapota leaf methanol extract at 12 24 and48 120583gmL of Manilkara zapota leaf methanol extract for 24 48 and 72 h and observed under an inverted light microscope (Magnification400times)The cells showed the typical characteristics of apoptosis such as cellular shrinkage (CS) apoptotic bodies (AB) nuclear fragmentation(NF) nuclear compaction (NC) and membrane blebbing (MB)

of the antioxidant enzyme which serves as a safeguard forcells during ROS detoxification [31] This result indicatesthat untreated cells exhibited a reduction of catalase levelassociated with a reduction of antioxidative capacity Con-versely the catalase levels in the treatment groups [12 120583gmL(857 plusmn 003 nmol H2O2 consumed minminus1 mgminus1 protein)24 120583gmL (1029 plusmn 001 nmol H2O2 consumed minminus1 mgminus1protein) and 48 120583gmL of Manilkara zapota leaf methanolextract (908 plusmn 004 nmol H2O2 consumed minminus1 mgminus1protein)] were significantly increased compared with that ofthe control group (694 plusmn 001 nmol H2O2 consumed minminus1mgminus1 protein) (P lt 005)

Numerous anticancer agents induce apoptotic cell deathvia induction of oxidative stress to a threshold that compro-mises the cell proliferation thus resulting in an imbalancebetween antioxidant and ROS within cancer cells [32] Ourpresent study found that exposure to Manilkara zapota leafmethanol extract increased catalase activity It is proposedthat the antioxidant defense system in HeLa cells is activatedin response to the accumulation of cellular oxidative stressproduced by Manilkara zapota leaf methanol extract Cata-lase is a vital endogenous antioxidant enzyme that detoxifieshydrogen peroxide to water and oxygen thereby limiting theadverse effects of ROS [33] Interestingly plants commonlyexerted antioxidant activity in which some of them are

demonstrated to have distinguished apoptosis-inducing abil-ity via induction of oxidative stress [34 35] In this contextcatalase was elevated in order to scavenge the ROS gener-ation induced by Manilkara zapota leaf methanol extractCollectively our results suggest that ROS level induced by theextract was high and has surpassed the antioxidant capacitythus resulting in apoptosis in HeLa cells

310Manilkara Zapota LeafMethanol Extract Triggers Releaseof Cytochrome c To further investigate the mechanism ofManilkara zapota leaf methanol extract induced apoptosisin HeLa cells we evaluated the transcriptional activity ofcytochrome c using real-time polymerase chain reaction(PCR) The cells were exposed to different concentrationsof Manilkara zapota leaf methanol extract (12 24 and48 120583gmL) and the release of cytochrome c was assessedAs shown in Figure 9 treatment with 24 and 48 120583gmLof Manilkara zapota leaf methanol extract resulted in theelevation of cytochrome c mRNA level An earlier studyhas demonstrated that translocation of Bax to mitochondriacan induce the outer mitochondrial membrane potentialand thus release of cytochrome c to the cytosol [36] whichactivates caspase cascade and cause apoptotic cell deathIn the present study it is conceivable that sufficient Baxappears to reside at the mitochondrial membrane to trigger


(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M

12 24 480Concentration (gmL)

01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)

Figure 4 Assessment of cell cycle kinetics in (a) untreated HeLa cells and HeLa cells treated withManilkara zapota leaf methanol extract atconcentrations of (b) 12 120583gmL (c) 24 120583gmL and (d) 48 120583gmL for 72 h and the cell cycle kinetic was determined by flow cytometry (e)The cell cycle analysis was determined using propidium iodide (PI) staining and analyzed by flow cytometry Values are reported as mean plusmnSD (n = 3) Value with different superscript letter indicates significant difference between groups by Tukeyrsquos test (P lt 005)


(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a

Early apoptoticLate apoptotic

Total apoptoticNecrosis


0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)

Figure 5 Evaluation ofManilkara zapota leafmethanol extract-induced apoptotic cell death in (a) untreatedHeLa cells andHeLa cells treatedwith (b) 12 120583gmL (c) 24 120583gmL and (d) 48 120583gmL ofManilkara zapota leaf methanol extract for 72 h (e) Assessment of apoptotic cell deathtreated withManilkara zapota leaf methanol extract was determined using Annexin V-FITC and propidium iodide (PI) staining assay usingflow cytometry Values are reported as mean plusmn SD (n = 3) Value with different superscript letter indicates significant difference betweengroups by Tukeyrsquos test (P lt 005)


a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b

12 24 48ControlConcentration (gmL)

0010203040506

Opt

ical

den

sity

(405

nm

)

(c)

Figure 6 Apoptotic activities of Manilkara zapota leaf methanol extract on HeLa cells after 72 h incubation HeLa cells treated with 12and 48 120583gmL of Manilkara zapota leaf methanol extract upregulated apoptotic protein expression of (a) Bax and downregulated (b) Bcl-2respectively (c)Manilkara zapota leaf methanol extract-induced caspase-3 activation Values are reported as mean plusmn SD (n = 3) Value withdifferent superscript letter indicates significant difference between groups by Tukeyrsquos test (P lt 005)

Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)

12 gmL 24 gmL 48 gmL

Figure 7Manilkara zapota leaf methanol extract induces cell apoptosis involved in reactive oxygen species (ROS) production in HeLa cellsThe levels of ROS were determined with DCFH-DA staining by flow cytometry after 72 h treatment with Manilkara zapota leaf methanolextract Values are reported as mean plusmn SD (n = 3) Value with different superscript letter indicates significant difference between groups byTukeyrsquos test (P lt 005)

cytochrome c release after Manilkara zapota leaf methanolextract treatment One of the predominant consequencesof mitochondrial cytochrome c release is the activation ofcaspase-3 Among the family of caspases caspase-3 has beendemonstrated as the most often triggered caspase protease inapoptotic cells which implies its critical role in the apoptoticcell death [37] Based on the findings caspase-3 was activatedafter treatment with Manilkara zapota leaf methanol extracttreatment and thus triggers the release of cytochrome csuggesting a caspase-dependent signal transduction pathway

311 Manilkara Zapota Leaf Methanol Extract Inhibits Acti-vation of EGFR in HeLa Cells Although growth factor-induced epidermal growth factor receptor (EGFR) signalingis required for several morphogenic processes and involvedin many cellular responses the deregulation of EGFR hasbeen associated with the proliferation and development ofcervical cancer [38] To gain a better understanding inwhich the Manilkara zapota leaf methanol extract inducesapoptosis we checked the changes of the transcriptionalactivity of EGFR following exposure ofManilkara zapota leaf

Evidence-Based Complementary and Alternative Medicine 15FL

2-H

P4 P4P4 P4

a

b b b

Control 12 gmL 24 gmL 48 gmL

0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H

Figure 8Manilkara zapota leaf methanol extract induced loss of mitochondria membrane potential HeLa cells were incubated with 12 24and 48 120583gmL ofManilkara zapota leaf methanol extract for 72 h and staining withMitoLite OrangeThe fluorescence intensity wasmeasuredusing NovoCyte Flow Cytometer with NovoExpress software Values are reported as mean plusmn SD (n = 3) Value with different superscript letterindicates significant difference between groups by Tukeyrsquos test (P lt 005)

a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n

Cytochrome cEGFRNF-B

Figure 9 mRNA levels of cytochrome c nuclear factor-kappa B (NF-120581B) and epidermal growth factor receptor (EGFR) in HeLa cells treatedwith Manilkara zapota leaf methanol extract for 72 h and evaluated using quantitative real-time polymerase chain reaction (PCR) Valuesare reported as mean plusmn SD (n = 3) Value with different superscript letter indicates significant difference between groups by Tukeyrsquos test (P lt005)


y = 01161x - 00957

00102030405060708

Abs

orba

nce

05 1 15 2 25 50Concentration (mgmL)

R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)

24 4812Concentration (gmL)

(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)

Figure 10 Standard curves of (a) gallic acid and (c) quercetin Total phenolic (b) and total flavonoid (d) contents in three differentconcentrations (12 24 and 48 120583gmL) of Manilkara zapota leaf methanol extract Values are reported as mean plusmn SD (n = 3) Value withdifferent superscript letter indicates significant difference between groups by Tukeyrsquos test (P lt 005)

methanol extract (12 24 and 48 120583gmL) on HeLa cells usingquantitative real-time PCR As illustrated in Figure 9 ourdata showed that untreated HeLa cells had the highest EGFRmRNA levels EGFR expression was significantly reducedin HeLa cells treated with Manilkara zapota leaf methanolextract compared to the untreated cells (P lt 005) Thesedata revealed that treatment with Manilkara zapota leafmethanol extract could diminish the EGFR activation withthe maximum effect observed at a concentration of 48120583gmL Manilkara zapota leaf methanol extract In additionto the effects observed in EGFR activity the role of NF-120581B in the inhibition of HeLa cells elicited by Manilkarazapota leaf methanol extract remains elusive Therefore wefurther explored the chemoprevention mechanism of NF-120581B on Manilkara zapota leaf methanol extract in HeLacells

312 Manilkara Zapota Leaf Methanol Extract DiminishesNF-120581B in HeLa Cells Nuclear factor-kappa B (NF-120581B) hasbeen linked with chronic inflammation and cancer [39]A study demonstrated that constitutively activation of NF-120581B is associated with cervical cancer [40] Hence the geneexpression level of NF-120581B in response to different concen-trations of Manilkara zapota leaf methanol extract (12 24and 48 120583gmL) was assessed in HeLa cells The NF-120581B familyis a group of inducible transcriptions which is involved ininflammatory and immune responses and thereby inhibited

cell apoptosis An earlier study demonstrated that cancer cellswith activated NF-120581B are resistant to ionizing radiation andchemotherapeutics Conversely inhibition of NF-120581B activitymarkedly elevates the sensitivity of cells to chemotherapeuticagents [41] The overall analysis indicated that untreatedHeLa cells presented the highest NF-120581B expression com-pared with the groups treated with Manilkara zapota leafmethanol extract InManilkara zapota leaf methanol extracttreated groups the phosphorylation and degradation of NF-120581B expression were increased in a dose-dependent manner(Figure 9) The suppression of NF-120581B transcriptional activ-ity resulting from the treatment of Manilkara zapota leafmethanol extract was consistent with the study reported byTan et al [42] who showed that brewersrsquo rice inhibited NF-120581B expression and thereby activated anti-inflammatory andantioxidant responses Collectively the findings presented inthis study suggest thatManilkara zapota leafmethanol extractmay modulate the inhibitory activity of HeLa cells via NF-120581Bsignaling

Most studies have demonstrated the synergistic andoradditive protective effects of several constituents [43] Wespeculated that this could be partially due to the polyphenoliccomponents which synergistically contribute to this antipro-liferative effect and apoptosis induction Furthermore wealso found that Manilkara zapota leaf methanol extract con-tains total phenolic (4255 plusmn 515 mg GAEg) total flavonoid(1160 plusmn 212 mg QE100 g) (Figure 10) and antioxidant


Table 3 Antioxidant activity and bioactive components in different concentrations ofManilkara zapota leaf methanol extract

Antioxidant activity andbioactive components 12 120583gmL 24 120583gmL 48 120583gmL

Beta-carotene bleachingtest () 258 plusmn 078a 515 plusmn 122b 1031 plusmn 142c

11-diphenyl-2-picryl-hydrazyl (DPPH)(mgmL)

0010 plusmn 0005a 0020 plusmn 0001b 0050 plusmn 0003c

Caffeic acid (120583gg) 00005 plusmn 00001a 00010 plusmn 00003b 00019 plusmn 00002c

Vanillic acid (120583gg) 0010 plusmn 0002a 0020 plusmn 0001b 0050 plusmn 0004c

p-coumaric acid (120583gg) 011 plusmn 005a 021 plusmn 001b 043 plusmn 012c

Ferulic acid (120583gg) 095 plusmn 012a 190 plusmn 009b 380 plusmn 011c

Values are reported as mean plusmn SD (n = 3) Value with different superscript letter in the same row indicates significant difference between groups by Tukeyrsquos test(P lt 005)

activity as determined using 120573-carotene bleaching test (4294plusmn 373) and 11-diphenyl-2-picryl-hydrazyl (DPPH) radicalscavenging capacity (048 plusmn 001 mgmL) and phenolic com-pounds [mainly caffeic acid (004 plusmn 001 120583gg) vanillic acid(093 plusmn 001 120583gg) p-coumaric acid (089 plusmn 016 120583gg) andferulic acid (7924 plusmn 1595 120583gg)] (unpublished data) Table 3shows the antioxidant activity and bioactive components indifferent concentrations of Manilkara zapota leaf methanolextract

Phytochemical screening is one of the methods that havebeen employed to evaluate the antioxidant constituents in aplant sample There are three categories of plant chemicalsnamely phenolic metabolites terpenoids and alkaloids [44]Of these plant chemicals phenolic compounds are the mostcritical for dietary applications due to an inverse associatedwith chronic diseases [45] Emerging evidence had provedtheir protective activity against human diseases [46 47] Ofall phytochemicals only saponins are present in Manilkarazapota leaf methanol extract None of the steroids triter-penoids flavonoids and phlobatannins is detected in theextract In support of these findings previous qualitativeanalysis of Manilkara zapota seed methanol extract hasexhibited the presence of saponins glycosides and phenols[48] Taken together the observed apoptotic effect and reduc-tion of EGFR and NF-120581B transcriptional activities are likelyattributed to the synergisticadditive effects of the phenoliccompounds and antioxidant activity present in Manilkarazapota leaf methanol extract

4 Conclusions

Our data suggest thatManilkara zapota leaf methanol extractmay have extensive application as an anticervical canceragent Importantly this extract is nontoxic with regard to thecell proliferation of mouse fibroblast (BALBc 3T3) cell lineTreatment withManilkara zapota leaf methanol extract led toa collapsed mitochondrial membrane potential which subse-quently triggered the release of cytochrome c thus leading tothe caspase cascade and ultimately resulting in the activationof the mitochondrial pathway which may play a vital role inthe apoptosis Additionally the decreased viability of HeLacells via induction of apoptosis and reduction of EGFR and

NF-120581B transcriptional activities is expected to leadManilkarazapota leaf methanol extract on target epithelial cells tosuppress the proliferation of cancerous lesions in the contextof cancer chemoprevention However further studies arewarranted to evaluate the anticancer activity of Manilkarazapota leaf methanol extract in animal models in orderto provide valuable insights to develop it as a therapeuticapproach for the treatment of human cervical cancer

Data Availability

All the data are contained within the manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Bee Ling Tan designed the study conducted the lab work wasresponsible for data acquisition and performed data analysisand interpretation and wrote the manuscript Mohd EsaNorhaizan advised lab work and commented on the finalversion of themanuscript LeeChinChanparticipated in flowcytometry analysis

Acknowledgments

The authors would like to thank Ministry of Science Tech-nology and Innovation (MOSTI) Malaysia (Project no 02-01-04-SF2141) for financial support

References

[1] World Health Organization Cervical Cancer 2017 httpwwwwhointcancerpreventiondiagnosis-screeningcervical-canceren

[2] K Tewari and B Monk ldquoInvasive cervical cancerrdquo in ClinicalGynecologic Oncology P Disaia and W Creasman Eds pp 51ndash119 Mosby Philadelphia Pa USA 2012


[3] J Wykosky T Fenton F Furnari and W K Cavenee ldquoThera-peutic targeting of epidermal growth factor receptor in humancancer Successes and limitationsrdquo Chinese Journal of Cancervol 30 no 1 pp 5ndash12 2011

[4] Y Xia S Shen and I M Verma ldquoNF-120581B an active player inhuman cancersrdquo Cancer Immunology Research vol 2 no 9 pp823ndash830 2014

[5] S ETHurđevic K Savikin J Zivkovic et al ldquoAntioxidant andcytotoxic activity of fatty oil isolated by supercritical fluidextraction from microwave pretreated seeds of wild growingPunica granatum LrdquoThe Journal of Supercritical Fluids vol 133pp 225ndash232 2018

[6] CMazewski K Liang and E Gonzalez deMejia ldquoComparisonof the effect of chemical composition of anthocyanin-rich plantextracts on colon cancer cell proliferation and their potentialmechanism of action using in vitro in silico and biochemicalassaysrdquo Food Chemistry vol 242 pp 378ndash388 2018

[7] Q D Do A E Angkawijaya P L Tran-Nguyen et al ldquoEffectof extraction solvent on total phenol content total flavonoidcontent and antioxidant activity of Limnophila aromaticrdquoJournal of Food and Drug Analysis vol 22 no 3 pp 296ndash3022014

[8] C Chen L Wang R Wang et al ldquoPhenolic contents cellularantioxidant activity and antiproliferative capacity of differentvarieties of oatsrdquo Food Chemistry vol 239 pp 260ndash267 2018

[9] Z L Ke Y pan X Xu C Nie and Z Zhou ldquoCitrus flavonoidsand human cancersrdquo Journal of Food and Nutrition Researchvol 3 no 5 pp 341ndash351 2015

[10] P Rajendran N Nandakumar T Rengarajan et al ldquoAntioxi-dants and human diseasesrdquo Clinica Chimica Acta vol 436 pp332ndash347 2014

[11] A Ghani Medicinal Plants of Bangladesh Chemical Con-stituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 2003

[12] M Y HajiMohiddinW Chin andDHoldsworth ldquoTraditionalmedicinal plants of Brunei Darussalam part III SengkurongrdquoInternational Journal of Pharmacognosy vol 30 no 2 pp 105ndash108 1992

[13] B L Tan M E Norhaizan H J Suhaniza C C LaiS Norazalina and K Roselina ldquoAntioxidant properties andantiproliferative effect of brewersrsquo rice extract (temukut) onselected cancer cell linesrdquo International Food Research Journalvol 20 no 5 pp 2117ndash2124 2013

[14] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[15] A Meda C E Lamien M Romito J Millogo and O GNacoulma ldquoDetermination of the total phenolic flavonoid andproline contents in Burkina Fasan honey as well as their radicalscavenging activityrdquo Food Chemistry vol 91 no 3 pp 571ndash5772005

[16] K Shanmugapriya P S Saravana H Payal S PeerMohammedand W Binnie ldquoAntioxidant activity total phenolic andflavonoid contents of Artocarpus heterophyllus and Manilkarazapota seeds and its reduction potentialrdquo International Journalof Pharmacy and Pharmaceutical Sciences vol 3 no 5 pp 256ndash260 2011

[17] D Zhang and Y Hamauzu ldquoPhenolics ascorbic acidcarotenoids and antioxidant activity of broccoli and theirchanges during conventional and microwave cookingrdquo FoodChemistry vol 88 no 4 pp 503ndash509 2004

[18] B L Tan M E Norhaizan S K Yeap and K RoselinaldquoWater extract of Brewersrsquo rice induces antiproliferation of

human colorectal cancer (HT-29) cell lines via the induction ofapoptosisrdquo European Review for Medical and PharmacologicalSciences vol 19 no 6 pp 1022ndash1029 2015

[19] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1973

[20] W C EvansTreasae and Evans Pharmacognosy Harcourt Braceand company Asia Pvt Ltd Singapore 1997

[21] J Dai and R J Mumper ldquoPlant phenolics extraction analysisand their antioxidant and anticancer propertiesrdquoMolecules vol15 no 10 pp 7313ndash7352 2010

[22] P Prayong S Barusrux and N Weerapreeyakul ldquoCytotoxicactivity screening of some indigenous Thai plantsrdquo Fitoterapiavol 79 no 7-8 pp 598ndash601 2008

[23] N Zhang T Bing X Liu et al ldquoCytotoxicity of guanine-based degradation products contributes to the antiproliferativeactivity of guanine-rich oligonucleotidesrdquoChemical Science vol6 no 7 pp 3831ndash3838 2015

[24] T A Bhat A K Chaudhary S Kumar et al ldquoEndoplasmicreticulum-mediated unfolded protein response and mitochon-drial apoptosis in cancerrdquo Biochimica et Biophysica Acta (BBA)- Reviews on Cancer vol 1867 no 1 pp 58ndash66 2017

[25] Z-Z Zou P-P Nie Y-W Li et al ldquoSynergistic inductionof apoptosis by salinomycin and gefitinib through lysosomaland mitochondrial dependent pathway overcomes gefitinibresistance in colorectal cancerrdquo Oncotarget vol 8 no 14 pp22414ndash22432 2017

[26] Y-C Hsu J-H Chiang C-S Yu et al ldquoAntitumor effects ofdeguelin on H460 human lung cancer cells in vitro and invivo Roles of apoptotic cell death and H460 tumor xenograftsmodelrdquo Environmental Toxicology vol 32 no 1 pp 84ndash98 2017

[27] Y Zhu T Tchkonia H Fuhrmann-Stroissnigg et al ldquoIdentifi-cation of a novel senolytic agent navitoclax targeting the Bcl-2family of anti-apoptotic factorsrdquo Aging Cell vol 15 no 3 pp428ndash435 2016

[28] W Nieves-Neira and Y Pommier ldquoApoptotic response to camp-tothecin and 7-hydroxystaurosporine (UCN-01) in the 8 humanbreast cancer cell lines of the NCI anticancer drug screen mul-tifactorial relationships with topoisomerase I protein kinaseC Bcl-2 p53 MDM-2 and caspase pathwaysrdquo InternationalJournal of Cancer vol 82 no 3 pp 396ndash404 1999

[29] V J Victorino L Pizzatti P Michelletti and C Panis ldquoOxida-tive stress redox signaling and cancer chemoresistance puttingtogether the pieces of the puzzlerdquo Current Medicinal Chemistryvol 21 no 28 pp 3211ndash3226 2014

[30] A Tsuchiya Y Kaku T Nakano and T Nishizaki ldquoDiarachi-donoylphosphoethanolamine induces apoptosis of malignantpleural mesothelioma cells through a TrxASK1p38 MAPKpathwayrdquo Journal of Pharmacological Sciences vol 129 no 3 pp160ndash168 2015

[31] J Du D He L-N Sun et al ldquoSemen Hoveniae extractprotects against acute alcohol-induced liver injury in micerdquoPharmaceutical Biology vol 48 no 8 pp 953ndash958 2010

[32] X Zou J Liang J Sun et al ldquoAllicin sensitizes hepatocellularcancer cells to anti-tumor activity of 5-fluorouracil throughROS-mediated mitochondrial pathwayrdquo Journal of Pharmaco-logical Sciences vol 131 no 4 pp 233ndash240 2016

[33] M M Goyal and A Basak ldquoHuman catalase Looking forcomplete identityrdquo Protein amp Cell vol 1 no 10 pp 888ndash8972010

[34] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolic


extract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[35] O S Aydos A Avci T Ozkan et al ldquoAntiproliferative apoptoticand antioxidant activities of wheatgrass (Triticum aestivum L)extract on CML (K562) cell linerdquo Turkish Journal of MedicalSciences vol 41 no 4 pp 657ndash663 2011

[36] I S Goping A Gross J N Lavoie et al ldquoRegulated targeting ofBAX to mitochondriardquoThe Journal of Cell Biology vol 143 no1 pp 207ndash215 1998

[37] H Kim H Choi and S K Lee ldquoEpstein-Barr virus microRNAmiR-BART20-5p suppresses lytic induction by inhibiting BAD-mediated caspase-3-dependent apoptosisrdquo Journal of Virologyvol 90 no 3 pp 1359ndash1368 2016

[38] R Narayanan H N Kim N K Narayanan D Nargi andB A Narayanan ldquoEpidermal growth factor-stimulated humancervical cancer cell growth is associated with EGFR and cyclinD1 activation independent of COX-2 expression levelsrdquo Inter-national Journal of Oncology vol 40 no 1 pp 13ndash20 2012

[39] A Korniluk O Koper H Kemona and V Dymicka-PiekarskaldquoFrom inflammation to cancerrdquo Irish Journal of Medical Sciencevol 186 no 1 pp 57ndash62 2017

[40] J Zhang H Wu P Li Y Zhao M Liu and H Tang ldquoNF-120581B-modulatedmiR-130a targets TNF-120572 in cervical cancer cellsrdquoJournal of Translational Medicine vol 12 no 1 p 155 2014

[41] C-Y Wang M W Mayo and A S Baldwin Jr ldquoTNF- andcancer therapy-induced apoptosis potentiation by inhibition ofNF-120581Brdquo Science vol 274 no 5288 pp 784ndash787 1996

[42] B L Tan M E Norhaizan K Huynh S K Yeap H Hazilawatiand K Roselina ldquoBrewersrsquo rice modulates oxidative stress inazoxymethane-mediated colon carcinogenesis in ratsrdquo WorldJournal of Gastroenterology vol 21 no 29 pp 8826ndash8835 2015

[43] B L Tan M E Norhaizan K Huynh et al ldquoWater extract ofbrewersrsquo rice induces apoptosis in human colorectal cancer cellsvia activation of caspase-3 and caspase-8 and downregulates theWnt120573-catenin downstream signaling pathway in brewersrsquo rice-treated rats with azoxymethane-induced colon carcinogenesisrdquoBMC Complementary and Alternative Medicine vol 15 p 2052015

[44] S K Santhosh A Venugopal andM C Radhakrishnan ldquoStudyon the phytochemicals antibacterial and antioxidant activitiesof Simarouba glaucardquo South Indian Journal of Biological Sci-ences vol 2 no 1 pp 119ndash124 2016

[45] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016

[46] I A Freires S M De Alencar and P L Rosalen ldquoA pharmaco-logical perspective on the use of Brazilian Red Propolis and itsisolated compounds against human diseasesrdquo European Journalof Medicinal Chemistry vol 110 pp 267ndash279 2016

[47] A I Hamed R B Said B Kontek et al ldquoLC-ESI-MSMSprofile of phenolic and glucosinolate compounds in samh flour(Mesembryanthemum forsskalei Hochst ex Boiss) and theinhibition of oxidative stress by these compounds in humanplasmardquo Food Research International vol 85 pp 282ndash290 2016

[48] C Mohanapriya S Uma R D Modilal and V NithyalakshmildquoPhytochemical screening and in vitro antioxidant studies onacetone extract of Manilkara zapota L seedsrdquo InternationalJournal of Pharmaceutical Sciences and Research vol 5 no 6pp 2354ndash2361 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


and inhibit apoptosis but it also facilitates metastasis [4]Given the pivotal role of NF-120581B in human cancer initiationdevelopment and metastasis NF-120581B pathway may serve as apromising therapeutic target

There have been extensive studies on natural compoundswhich showed a potent antiproliferative activity in con-junction with having good antioxidant activities [5 6]Many plants especially traditional medicinal plants havebeen widely investigated for their antioxidant activity inthe last few decades [7] Emerging evidence has demon-strated the role of natural antioxidant in the prevention ofchronic diseases including cancer and inflammation [8ndash10]In continuation of the efforts towards the discovery of bettertreatment strategies for cervical cancer plants have gainedremarkable interest as an effective anticancer agent In linewith this there is an unmet need to discover new anticanceragent with high efficacy and specificity but showing minimaladverse outcome

Manilkara zapota (L) P Royen (family Sapotaceae)commonly called sapodilla or locally known as ciku isan evergreen tree grown abundantly throughout India sub-continent including Bangladesh [11] though it is native toCentral America and Mexico Manilkara zapota leaf hasbeen traditionally used for the treatment of diarrhea coldand coughs [12] Nonetheless there is no pharmacologicalstudy on anticervical cancer properties of Manilkara zapotaleaf methanol extract in the literature Our earlier studydemonstrated that Manilkara zapota leaf water extract hascytotoxic activity towards human hepatocellular carcinoma(HepG2) and human colorectal adenocarcinoma (HT-29) celllines (unpublished data) Therefore this study was designedto investigate the ability to induce apoptosis and the under-lying mechanisms ofManilkara zapota leaf methanol extractinducing cytotoxicity in HeLa cells These molecular interac-tions underlying the apoptotic mediated signaling pathwayin cellular function may be involved in the modulation ofcervical cancer and deserve further elucidation

2 Materials And Methods

21 Chemicals and Reagents RPMI-1640 mediumMycoplex fetal bovine serum (FBS) penicillin andstreptomycin (100times) Dulbeccorsquos Modified Eagle Medium(DMEM) and trypsin-ethylenediaminetetraacetic acid(EDTA) (1times) were bought from Gibco (Grand IslandNY USA) Cycle TEST PLUS DNA Reagent Kit andAnnexin V-FITC Apoptosis Detection Kit I were procuredfrom BD Biosciences Pharmingen (Franklin Lakes NJUSA) Mitochondrial Membrane Potential Assay Kit(orange fluorescence) was bought from Abnova (Taipei CityTaiwan) Bax and Bcl-2 Human SimpleStep ELISAKits wereobtained from Abcam UK Caspase Colorimetric Assay Kitwas bought fromRampD Systems (MinneapolisMN USA) Allother reagents and chemicals used were of analytical gradeand obtained from Sigma-Aldrich (St Louis MO USA)

22 Plant Materials The plant (Manilkara zapota (L) PRoyen) was collected from Pahang Malaysia The plant

with voucher specimen number SK 317917 was deposited inBiodiversity Unit of Institute of Bioscience Universiti PutraMalaysia

23 Preparation of Plant Extract Initially leaf of Manilkarazapota was cut into small pieces and dried in an oven at40∘C for 3 days before being ground into powder formManilkara zapota leaf sample was extracted with methanolfollowing the method of Tan et al [13] About 5 g ofsamples was extracted with 40 mL of methanol in a shaker(Heidolph Inkubatorhaube Germany) at 40∘C for 2 h Theslurry was filtered using filter paper (Whatman no 1) Thefiltrate from the methanol extract was dried using rotaryevaporator (Buchi Rotavapor R-200 Switzerland) The yieldwas measured using electronic balance (Shimadzu KyotoJapan) and stored at -20∘C until further analyses The yieldof plant extract was calculated as follows

Percentage of plant yield ()

=Weight of plant extract (g)Weight of plant sample (g)

times 100(1)

24 Cell Culture The human colon carcinoma (HCT-116)human colorectal adenocarcinoma (HT-29) human cervicalcancer (HeLa) human hepatocellular carcinoma (HepG2)human gastric adenocarcinoma (HGT-1) human prostatecancer (PC-3) and mouse fibroblast (BALBc 3T3) celllines were obtained from American Type Culture Collection(ATCC Rockville MD USA) The HT-29 HCT-116 andHGT-1 cells were grown in DMEM supplemented with 100IUmL penicillin 100 120583gmL streptomycin and 10 (vv)FBSHeLaHepG2 PC-3 andBALBc 3T3 cells were culturedin RPMI-1640 medium supplemented with 100 IUmL peni-cillin 100 120583gmL streptomycin and 10 (vv) FBS All celllines were grown at 5CO2 atmosphere and 37∘Chumidifiedatmosphere incubator

25 Determination of Cell Viability using 3-(45-Dimethyl-thiazol-2-yl)-25-diphenyltetrazolium Bromide (MTT) AssayThe cell viability of HT-29 HCT-116 HeLa HGT-1 HepG2PC-3 and BALBc 3T3 upon treatment with Manilkarazapota leaf methanol extract was determined using MTTassay [13] HT-29 HCT-116 HeLa HGT-1 HepG2 PC-3 andBALBc 3T3 cells were seeded at a density of 5 times 104 cellswellin a 96-well plate After an overnight incubation the cellswere exposed to leaf methanol extract of Manilkara zapota(156-200 120583gmL) Untreated BALBc 3T3 and cancer celllines were included Following 24 48 and 72 h of treatment20 120583L of MTT (5 mgmL) was added to each well followedby 2-4 h incubation Lastly the media from each well werediscarded and 100 120583L of dimethyl sulfoxide (DMSO) wasadded to solubilize the purple-blue formazanThe absorbancewas read at 570 nm using an ELISA microplate reader(Tecan Switzerland) and 630 nm was used as a referencewavelength A graph of percentage of cell viability versusconcentration of Manilkara zapota leaf methanol extractwas plotted and the concentration of Manilkara zapota leafmethanol extract which inhibited 50 of cellular growth




times 100


(2)






























= C



(3)

















Table2Treatm

ento

fManilkarazapota

leafmethano

lextract(156-200120583g

mL)

onselected

cancer

celllin

esfor2

448and

72hevaluatedby

MTT

andLD

Hassays

Cancer

celllin

esMTT

(120583gmL)

LDH(120583gmL)

24h

48h

72h

24h

48h

72h

HT-29

9327plusmn1719

a8929plusmn601

a6912plusmn810

b9033plusmn1579a

8599plusmn487

a7622plusmn539

b

HCT

-116

9014plusmn1423a

8733plusmn92

9a8317plusmn99

2a9322plusmn90

3a9012plusmn97

7a8811plusmn1169a

HeLa

8929plusmn1820a

5923plusmn1033

a2387plusmn502

b8733plusmn1498a

8044plusmn1165a

2576plusmn893

b

HGT-1

8011plusmn1019

a7204plusmn523

a4944plusmn1062b

6520plusmn1427a

6211plusmn629

a5989plusmn1027a

HepG2

9729plusmn326

a8395plusmn92

0ab

7302plusmn93

3b8945plusmn1682a

8303plusmn535

a7704plusmn99

3a

HCT

-116h

uman

coloncarcinom

aHeLa

human

cervical

cancerH

epG2

human

hepatocellu

larcarcinom

aHGT-1hu

man

gastr

icadenocarcino

ma

HT-29h

uman

colorectal

adenocarcino

ma

LDHlactate

dehydrogenaseandMTT

3-(45-dimethylth

iazol-2

-yl)-25-diph

enyltetrazolium

brom

ide

Values

arerepo

rted

asmeanplusmnSD

(n=3)V

alue

with

different



ifference

byTu

keyrsquos

test(Plt005)In

MTT

assaytre

atmentw

ithManilkarazapota

leafmethano

lextractfor7

2h(691


significantly

inhibitedthep

roliferationof

HT-29

cells

comparedto

24h(9327plusmn1719120583gmL)

(Plt005)whereasinLD

Hassaytherew

asa

significanteffectof

thecytotoxicactiv

ities

ofManilkarazapota

leafmethano

lextractin

HT-29

cells

incubatedfor7

2h(7622plusmn539120583gmL)

comparedto

thoseincubatedfor2

4h(9033plusmn1579120583gmL)

or48


(Plt005)









0 40 60 8020 160

180

100

120

200

140

Concentration (gmL)

010203040506070

Perc

enta

ge o

fvi

able

cells

()

(a)

aab ab ab ab b

c c

ab a ab abcbc

c

d d

aa a a a a

b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(b)

a a aa a

ab

bcc

aab abc

abc bc cdd

e

a a a ab b

b

c

24 h48 h72 h

25 50 100

200

625

125

156

3

312

5

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(c)

a a aa a a a a

a a a a a a a aa ab

ab ab ab ab b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(d)

24 h48 h72 h



25 50 200

100

125

625

312

5

156


020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(e)








hou

rs48

hou

rs24

hou

rs

50 m

50 m

50 m 50 m 50 m

50 m

50 m 50 m

50 m 50 m

50 m 50 m








50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs







(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















times 100


(2)






























= C



(3)

















Table2Treatm

ento

fManilkarazapota

leafmethano

lextract(156-200120583g

mL)

onselected

cancer

celllin

esfor2

448and

72hevaluatedby

MTT

andLD

Hassays

Cancer

celllin

esMTT

(120583gmL)

LDH(120583gmL)

24h

48h

72h

24h

48h

72h

HT-29

9327plusmn1719

a8929plusmn601

a6912plusmn810

b9033plusmn1579a

8599plusmn487

a7622plusmn539

b

HCT

-116

9014plusmn1423a

8733plusmn92

9a8317plusmn99

2a9322plusmn90

3a9012plusmn97

7a8811plusmn1169a

HeLa

8929plusmn1820a

5923plusmn1033

a2387plusmn502

b8733plusmn1498a

8044plusmn1165a

2576plusmn893

b

HGT-1

8011plusmn1019

a7204plusmn523

a4944plusmn1062b

6520plusmn1427a

6211plusmn629

a5989plusmn1027a

HepG2

9729plusmn326

a8395plusmn92

0ab

7302plusmn93

3b8945plusmn1682a

8303plusmn535

a7704plusmn99

3a

HCT

-116h

uman

coloncarcinom

aHeLa

human

cervical

cancerH

epG2

human

hepatocellu

larcarcinom

aHGT-1hu

man

gastr

icadenocarcino

ma

HT-29h

uman

colorectal

adenocarcino

ma

LDHlactate

dehydrogenaseandMTT

3-(45-dimethylth

iazol-2

-yl)-25-diph

enyltetrazolium

brom

ide

Values

arerepo

rted

asmeanplusmnSD

(n=3)V

alue

with

different



ifference

byTu

keyrsquos

test(Plt005)In

MTT

assaytre

atmentw

ithManilkarazapota

leafmethano

lextractfor7

2h(691


significantly

inhibitedthep

roliferationof

HT-29

cells

comparedto

24h(9327plusmn1719120583gmL)

(Plt005)whereasinLD

Hassaytherew

asa

significanteffectof

thecytotoxicactiv

ities

ofManilkarazapota

leafmethano

lextractin

HT-29

cells

incubatedfor7

2h(7622plusmn539120583gmL)

comparedto

thoseincubatedfor2

4h(9033plusmn1579120583gmL)

or48


(Plt005)









0 40 60 8020 160

180

100

120

200

140

Concentration (gmL)

010203040506070

Perc

enta

ge o

fvi

able

cells

()

(a)

aab ab ab ab b

c c

ab a ab abcbc

c

d d

aa a a a a

b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(b)

a a aa a

ab

bcc

aab abc

abc bc cdd

e

a a a ab b

b

c

24 h48 h72 h

25 50 100

200

625

125

156

3

312

5

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(c)

a a aa a a a a

a a a a a a a aa ab

ab ab ab ab b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(d)

24 h48 h72 h



25 50 200

100

125

625

312

5

156


020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(e)








hou

rs48

hou

rs24

hou

rs

50 m

50 m

50 m 50 m 50 m

50 m

50 m 50 m

50 m 50 m

50 m 50 m








50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs







(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of









































= C



(3)

















Table2Treatm

ento

fManilkarazapota

leafmethano

lextract(156-200120583g

mL)

onselected

cancer

celllin

esfor2

448and

72hevaluatedby

MTT

andLD

Hassays

Cancer

celllin

esMTT

(120583gmL)

LDH(120583gmL)

24h

48h

72h

24h

48h

72h

HT-29

9327plusmn1719

a8929plusmn601

a6912plusmn810

b9033plusmn1579a

8599plusmn487

a7622plusmn539

b

HCT

-116

9014plusmn1423a

8733plusmn92

9a8317plusmn99

2a9322plusmn90

3a9012plusmn97

7a8811plusmn1169a

HeLa

8929plusmn1820a

5923plusmn1033

a2387plusmn502

b8733plusmn1498a

8044plusmn1165a

2576plusmn893

b

HGT-1

8011plusmn1019

a7204plusmn523

a4944plusmn1062b

6520plusmn1427a

6211plusmn629

a5989plusmn1027a

HepG2

9729plusmn326

a8395plusmn92

0ab

7302plusmn93

3b8945plusmn1682a

8303plusmn535

a7704plusmn99

3a

HCT

-116h

uman

coloncarcinom

aHeLa

human

cervical

cancerH

epG2

human

hepatocellu

larcarcinom

aHGT-1hu

man

gastr

icadenocarcino

ma

HT-29h

uman

colorectal

adenocarcino

ma

LDHlactate

dehydrogenaseandMTT

3-(45-dimethylth

iazol-2

-yl)-25-diph

enyltetrazolium

brom

ide

Values

arerepo

rted

asmeanplusmnSD

(n=3)V

alue

with

different



ifference

byTu

keyrsquos

test(Plt005)In

MTT

assaytre

atmentw

ithManilkarazapota

leafmethano

lextractfor7

2h(691


significantly

inhibitedthep

roliferationof

HT-29

cells

comparedto

24h(9327plusmn1719120583gmL)

(Plt005)whereasinLD

Hassaytherew

asa

significanteffectof

thecytotoxicactiv

ities

ofManilkarazapota

leafmethano

lextractin

HT-29

cells

incubatedfor7

2h(7622plusmn539120583gmL)

comparedto

thoseincubatedfor2

4h(9033plusmn1579120583gmL)

or48


(Plt005)









0 40 60 8020 160

180

100

120

200

140

Concentration (gmL)

010203040506070

Perc

enta

ge o

fvi

able

cells

()

(a)

aab ab ab ab b

c c

ab a ab abcbc

c

d d

aa a a a a

b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(b)

a a aa a

ab

bcc

aab abc

abc bc cdd

e

a a a ab b

b

c

24 h48 h72 h

25 50 100

200

625

125

156

3

312

5

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(c)

a a aa a a a a

a a a a a a a aa ab

ab ab ab ab b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(d)

24 h48 h72 h



25 50 200

100

125

625

312

5

156


020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(e)








hou

rs48

hou

rs24

hou

rs

50 m

50 m

50 m 50 m 50 m

50 m

50 m 50 m

50 m 50 m

50 m 50 m








50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs







(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

































Table2Treatm

ento

fManilkarazapota

leafmethano

lextract(156-200120583g

mL)

onselected

cancer

celllin

esfor2

448and

72hevaluatedby

MTT

andLD

Hassays

Cancer

celllin

esMTT

(120583gmL)

LDH(120583gmL)

24h

48h

72h

24h

48h

72h

HT-29

9327plusmn1719

a8929plusmn601

a6912plusmn810

b9033plusmn1579a

8599plusmn487

a7622plusmn539

b

HCT

-116

9014plusmn1423a

8733plusmn92

9a8317plusmn99

2a9322plusmn90

3a9012plusmn97

7a8811plusmn1169a

HeLa

8929plusmn1820a

5923plusmn1033

a2387plusmn502

b8733plusmn1498a

8044plusmn1165a

2576plusmn893

b

HGT-1

8011plusmn1019

a7204plusmn523

a4944plusmn1062b

6520plusmn1427a

6211plusmn629

a5989plusmn1027a

HepG2

9729plusmn326

a8395plusmn92

0ab

7302plusmn93

3b8945plusmn1682a

8303plusmn535

a7704plusmn99

3a

HCT

-116h

uman

coloncarcinom

aHeLa

human

cervical

cancerH

epG2

human

hepatocellu

larcarcinom

aHGT-1hu

man

gastr

icadenocarcino

ma

HT-29h

uman

colorectal

adenocarcino

ma

LDHlactate

dehydrogenaseandMTT

3-(45-dimethylth

iazol-2

-yl)-25-diph

enyltetrazolium

brom

ide

Values

arerepo

rted

asmeanplusmnSD

(n=3)V

alue

with

different



ifference

byTu

keyrsquos

test(Plt005)In

MTT

assaytre

atmentw

ithManilkarazapota

leafmethano

lextractfor7

2h(691


significantly

inhibitedthep

roliferationof

HT-29

cells

comparedto

24h(9327plusmn1719120583gmL)

(Plt005)whereasinLD

Hassaytherew

asa

significanteffectof

thecytotoxicactiv

ities

ofManilkarazapota

leafmethano

lextractin

HT-29

cells

incubatedfor7

2h(7622plusmn539120583gmL)

comparedto

thoseincubatedfor2

4h(9033plusmn1579120583gmL)

or48


(Plt005)









0 40 60 8020 160

180

100

120

200

140

Concentration (gmL)

010203040506070

Perc

enta

ge o

fvi

able

cells

()

(a)

aab ab ab ab b

c c

ab a ab abcbc

c

d d

aa a a a a

b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(b)

a a aa a

ab

bcc

aab abc

abc bc cdd

e

a a a ab b

b

c

24 h48 h72 h

25 50 100

200

625

125

156

3

312

5

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(c)

a a aa a a a a

a a a a a a a aa ab

ab ab ab ab b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(d)

24 h48 h72 h



25 50 200

100

125

625

312

5

156


020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(e)








hou

rs48

hou

rs24

hou

rs

50 m

50 m

50 m 50 m 50 m

50 m

50 m 50 m

50 m 50 m

50 m 50 m








50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs







(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























0 40 60 8020 160

180

100

120

200

140

Concentration (gmL)

010203040506070

Perc

enta

ge o

fvi

able

cells

()

(a)

aab ab ab ab b

c c

ab a ab abcbc

c

d d

aa a a a a

b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(b)

a a aa a

ab

bcc

aab abc

abc bc cdd

e

a a a ab b

b

c

24 h48 h72 h

25 50 100

200

625

125

156

3

312

5

Concentration (gmL)

020406080

100120140160

Perc

enta

ge o

fvi

able

cells

()

(c)

a a aa a a a a

a a a a a a a aa ab

ab ab ab ab b b

24 h48 h72 h

5025 100

200

625

125

312

5

156

3

Concentration (gmL)

020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(d)

24 h48 h72 h



25 50 200

100

125

625

312

5

156


020406080

100120

Perc

enta

ge o

fvi

able

cells

()

(e)








hou

rs48

hou

rs24

hou

rs

50 m

50 m

50 m 50 m 50 m

50 m

50 m 50 m

50 m 50 m

50 m 50 m








50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs







(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















hou

rs48

hou

rs24

hou

rs

50 m

50 m

50 m 50 m 50 m

50 m

50 m 50 m

50 m 50 m

50 m 50 m








50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs







(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

50 m 50 m 50 m 50 m

AB

CS

CS

AB

MB

AB

CS

AB

CS

CS

MBNF

MB

AB

ABMB

CS NF

NF

NCNFNC


hou

rs48

hou

rs24

hou

rs







(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b)

(c) (d)

ab

b b

a

bc

b

c

a

b b ba

bc

bc

Sub-G0G0G1

SG2M


01020304050607080

Perc

enta

ge o

f cel

ls (

)

(e)



(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b)

(c) (d)

a a

b

c

a a a

b

a abb

c

a a a a




0

5

10

15

20

25

30

Perc

enta

ge o

f cel

ls (

)

(e)



a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















a

b

a a


0

10

20

30

40

50Ba

x (n

gm

g)

(a)

aab

ab

b


0

5

10

15

20

Bcl-2

(ng

mg)

(b)

ab

c

b


0010203040506

Opt

ical

den

sity

(405

nm

)

(c)


Control

FITC-H

Coun

t

a

b b

c

Intr

acel

lula

r Rea

ctiv

e


0

2

4

6

8

10

12

Oxy

gen

Spec

ies (

)






2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















2-H

P4 P4P4 P4

a

b b b


0

20

40

60

80

100

120

Perc

enta

ge m

ean

of P

4 (

)


FL1-H


a a

b b

a

bb

c

a

b

b

c


0

1

2

3

4

5

6

Rela

tive N

orm

aliz

ed E

xpre

ssio

n




y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















y = 01161x - 00957

00102030405060708

Abs

orba

nce


R2 = 09977

(a)

a

b

c

Tota

l phe

nolic

cont

ent

012345678

(mg

GA

Eg)


(b)

y = 00201x + 00912

0

005

01

015

02

025

Abs

orba

nce


R2 = 09986

(c)

a

b

c

Tota

l flav

onoi

d co

nten

t


0

20

40

60

80

100

120

140

(mg

QE

100

g)

(d)



















4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
































4 Conclusions



Data Availability






Acknowledgments


References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of









































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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