cytotoxic activities against breast cancer cells of local

Research ArticleCytotoxic Activities against Breast Cancer Cells of LocalJusticia gendarussa Crude Extracts

Zahidah Ayob1 Siti Pauliena Mohd Bohari1

Azman Abd Samad1 and Shajarahtunnur Jamil2

1Department of Biotechnology and Medical Engineering Faculty of Biosciences and Medical EngineeringUniversiti Teknologi Malaysia (UTM) 81310 Johor Bahru Malaysia2Department of Chemistry Faculty of Science Universiti Teknologi Malaysia (UTM) 81310 Johor Bahru Malaysia

Correspondence should be addressed to Siti Pauliena Mohd Bohari paulienautmmy

Received 14 August 2014 Revised 30 October 2014 Accepted 10 November 2014 Published 11 December 2014

Academic Editor Wagner Vilegas

Copyright copy 2014 Zahidah Ayob 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

Justicia gendarussa methanolic leaf extracts from five different locations in the Southern region of Peninsular Malaysia and twoflavonoids kaempferol and naringenin were tested for cytotoxic activity Kaempferol and naringenin were two flavonoids detectedin leaf extracts using gas chromatography-flame ionization detection (GC-FID) The results indicated that highest concentrationsof kaempferol and naringenin were detected in leaves extracted from Mersing with 159180mgkg and 44435mgkg respectivelyPositive correlationswere observed between kaempferol and naringenin concentrations in all leaf extracts analysedwith the Pearsonmethod The effects of kaempferol and naringenin from leaf extracts were examined on breast cancer cell lines (MDA-MB-231 andMDA-MB-468) using MTT assay Leaf extract from Mersing showed high cytotoxicity against MDA-MB-468 and MDA-MB-231with IC

50values of 23 120583gmL and 40 120583gmL respectively compared to other leaf extracts Kaempferol possessed high cytotoxicity

against MDA-MB-468 and MDA-MB-231 with IC50values of 23 120583gmL and 34 120583gmL respectively These findings suggest that the

presence of kaempferol in Mersing leaf extract contributed to high cytotoxicity of both MDA-MB-231 and MDA-MB-468 cancercell lines

1 Introduction

Breast cancer is the second largest cancer after lung cancerin the world and the most common malignancy amongwomen [1] In Malaysia the most frequent cancers are breastcancer (181) colorectal cancer (123) and lung cancer(102) these three cancers affect both women and men [2]Currently the most common approaches for treating humanbreast cancer include surgery radiotherapy hyperthermiahormone therapy and chemotherapy [3]

Breast cancers can be classified by stage pathologygrade and expression of oestrogen receptor (ER) proges-terone receptor (PR) or human epidermal growth factorreceptor (Her2neu) [4] The two types of breast cancercells that have gained interest among investigators andmedical research laboratories are MDA-MB-231 and MDA-MB-468 MDA-MB-231 cells are characterised as ER- PR-and Her2neu-negativebasal-B mammary carcinoma while

MDA-MB-468 cells are characterised as ER- PR- and Her2neu-negativebasal-A mammary carcinoma [4] MDA-MB-231 and MDA-MB-468 cells were derived from the pleuraleffusions of 51-year-old female patients MDA-MB-231 cellswere derived from a Caucasian female while MDA-MB-468cells were derived from an African American female [5ndash7]

There is strong social interest in natural remedies andmore than 80 of the world population considers traditionalmedicine as their source of primary health care [8] Indeedthere has been a worldwide effort to discover new anticanceragents from medicinal plants and various experimentalmodels of natural products have resulted in anticancer agents[9 10]

One of the potential medicinal plants that is beinginvestigated in our laboratory is J gendarussa which isalso known by its common name Gendarussa This plantis a member of the Acanthaceae family that can be foundubiquitously in many countries including Indonesia Sri

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 732980 12 pageshttpdxdoiorg1011552014732980

2 Evidence-Based Complementary and Alternative Medicine

Lanka India and Malaysia [11] The roots and leaf extractsof J gendarussa have been demonstrated to treat chronicrheumatism inflammation bronchitis headache arthri-tis vaginal discharges dyspepsia eye disease and fever[12]

Previous reports demonstrated that J gendarussa leafextracts have been used traditionally as a male contraceptiveagent by several ethnic groups in the central part of PapuaIndonesia This extract is able to inhibit mouse spermatozoapenetration of mice ovum [13] J gendarussa methanolicleaves and root extracts showed cytotoxic activity againstbrine shrimp in the brine shrimp lethality assay with IC

50

values of 4871 120583gmL and 9325 120583gmL respectively [14] Inaddition J gendarussa leaves and stem extracts were reportedto have anticancer antioxidant antibacterial antifungalantiangiogenic anthelmintic and hepatoprotective activities[15ndash23]

Phytochemical studies on leaves from J gendarussarevealed the presence of flavonoids alkaloids triterpenoidalsaponins amino acids aromatic amines stigmasterol andlupeol [18 24ndash27] Our previous study on green callus andin vitro leaf extracts of J gendarussa detected two flavonoidsthat is kaempferol and naringenin using GC-FID [28] Bothflavonoids were also detected in themethanolic leaf extract ofJ gendarussa using the same method [29] Bioactivity studieson both flavonoids found that it exhibited strong antioxidantand inhibitory effects on cholesterol in HepG2 cancer cells[30ndash32] Kaempferol also inhibited pancreatic cancer cell(MIAPaCa-2 and Panc-1) proliferation induced cancer cellapoptosis and prevented arteriosclerosis [30 33] Naringenindemonstrated cytotoxic effects against breast cancer cells(MCF-7) and suppressed apoptosis in mouse leukaemia P388cells [34ndash36] Our previous study on both flavonoids showedstrong cytotoxic activity against colonic (HT-29) cervical(HeLa) and pancreatic (BxPC-3) cells [29]

To the best of our knowledge this is the first study of theeffects of J gendarussa leaf extracts against human breast can-cer cell lines (MDA-MB-231 and MDA-MB-468) This studywas performed to screen the cytotoxic activities ofmethanolicleaf extracts from five different locations (Mersing MuarSkudai Batu Pahat and Pulai) in Johor and two flavonoids(naringenin and kaempferol) against breast cancer cell linesThe quantification of kaempferol and naringenin content inleaf extracts of J gendarussa using GC-FID was also carriedout

2 Methods and Materials

21 Plant Materials J gendarussa plants were collected fromfive different locations in Johor (Mersing Muar Skudai BatuPahat and Pulai) and maintained in a greenhouse at theFaculty of Biosciences and Medical Engineering UniversitiTeknologi Malaysia (UTM) The J gendarussa plant wasidentified byDr RichardChungChengKong senior researchofficer of the Forest Research Institute of Malaysia (FRIM)The voucher specimen (PID-100214-06) was deposited atHerbarium Management Branch Flora Biodiversity Pro-gram Forest Biodiversity Division FRIM Kepong SelangorMalaysia

22 General Chemicals Commercial standards (kaempferoland naringenin) were purchased from Sigma-Aldrich (Sub-ang Jaya Selangor Malaysia) Tamoxifen was used as apositive control in the MTT assay All samples were dilutedwith 01 of dimethylsulfoxide (DMSO) which has no effecton cell viability [37]

23 Preparation of Extracts The J gendarussa leaves were air-dried for 4 weeks The dried leaves were ground into smallparticles and approximately 50 g of small particles was soakedinto 1000mLofmethanol at room temperature for 72 hours ina ratio of 1 20 (wv) [10] The mixtures were filtered throughsterile cotton and filtered again using Whatman number 1filter paper to obtain methanolic supernatants The filteredmethanolic extract was evaporated at 40∘C under reducedpressure by using an EYELA N-1000 rotary evaporator(Bohemia NY USA)The dried crude extract was kept at 4∘Cprior to use

24 Quantification of Flavonoids in Leaf Extracts GC-FIDand quantitative analysis were performed according to previ-ously published method [38] GC-FID (HP-6890N AgilentUSA) equipped with a HP-5 fused silica capillary column(300m times 032mm ID times 025120583m) was used The temperatureprogrammed was 100∘C held for 1 minute and then rampedto 275∘C at 10∘Cmin and held for 17 minutes at 275∘C Theinjection temperature was 275∘C The flow rate of the carriergas (helium) was 1mLmin A split ratio of 50 1 was used Aquantity of 5120583L of leaf extract and standardswas injectedThechromatographic data were recorded and processed usingAgilent Cerity QA-QC software

25 Cell Culture MDA-MB-231 (basal-B mammary carci-noma) and MDA-MB-468 (basal-A mammary carcinoma)breast cancer cell lines and CHO (Chinese hamster ovary)normal cell line were obtained from American Type CultureCollection (ATCC) and as a generous gift from Dr Sale-hhuddin Hamdan (Animal Cell Culture Laboratory Facultyof Biosciences and Medical Engineering UTM) MDA-MB-231 and MDA-MB-468 breast cancer cell lines were culturedin Dulbeccorsquos modified Eaglersquos medium (DMEM) whileCHO normal cells were cultured in Roswell Park MemorialInstitute 1640 (RPMI-1640) medium supplemented with 10vv foetal bovine serum (FBS) 100UmL of penicillin and100 120583gmL of streptomycin as a complete growth mediumCells were maintained in 25 cm2 flasks and incubatedin a humidified incubator (CO

2Water-Jacketed Incubator

NuAire Fernbrook Lane Plymouth USA) at 37∘C with 5CO2 All materials were obtained from Gibco (Gibco Bio-

Diagnostics Petaling Jaya Selangor Malaysia)

26 MTTAssay Cytotoxicity testing was performed using 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT Sigma) according to the method reported in previousstudies [29 39] In this assay cells were harvested afterreaching 80 confluence Before starting theMTT assay cellswere optimised at different seeding densities ranging from20 times 103 cellmL to 10 times 106 cellmL in light to determine

Evidence-Based Complementary and Alternative Medicine 3

appropriate seeding number for the experiment Each well ofthe microtiter plate (96-well) was filled with 100120583L of cellsuspension (MDA-MB-231 MDA-MB-468 and CHO withthe seeding number 5 times 104 cellmL) in complete growthmedium After 24 hours of incubation cells were treatedwith leaf extracts of different concentrations ranging from781 to 1000120583gmL with a total well volume of 200120583L withtechnical replicates Microtiter plates were further incubatedfor 72 hours with plant extracts After 72 hours of incubation20120583L of MTT (a stock solution of 5mgmL in PBS) wasadded to each well and the plates incubated for 4 hoursat 37∘C Medium from each well was carefully removedwithout disturbing the MTT crystals in wells The MTTformazan crystals were dissolved by the addition of 1MHCl and 100mM isopropanol to each well After solubilisingthe purple formazan absorbance was measured using aBioRad microplate reader (Shinagawa-ku Tokyo Japan) ata wavelength of 575 nm Cytotoxic activity was recorded asIC50 which is the concentration necessary to reduce the

absorbance of treated cells by 50 compared to the control(untreated cells) [40]

27 Statistical Analysis All samples were run in three repli-cates Data obtained were analysed using SPSS softwarefor Windows (SPSS 160 for Windows Evaluation Versionsoftware SPSS Inc USA) The normality of the data wastested using the Shapiro-Wilk test The data were analysedusing the Independence 119905-test for normal data and Mann-Whitney 119880 test for nonnormal data The correlations wereanalysed using the Pearson correlation test [41] Differenceswere considered to achieve significance for probability 119875 lt005

3 Results

Phytochemical analysis of J gendarussa leaf extracts showedthat kaempferol and naringenin were quantified from fivedifferent locations byGC-FID Figure 1 shows the distributionof kaempferol and naringenin contents in leaf extracts

In this study cytotoxicity of J gendarussa leaf extractsfrom five different locations and flavonoids (kaempferolnaringenin and a mixture of kaempferol and naringenin)were tested against breast cancer cell lines (MDA-MB-231 andMDA-MB-468) and a normal cell line (CHO) using MTTassay Tamoxifen was used as a positive control The IC

50

values obtained referred to 50 of cells inhibited by plantextracts [42] In a previous study cytotoxicity was evaluatedbased on IC

50values where IC

50values below 20120583gmLwere

considered cytotoxic from 21 to 40120583gmL were consideredweak cytotoxic and above 40 120583gmL were not consideredcytotoxic [40 43 44]

Table 1 represents the IC50

values of J gendarussaleaf extracts flavonoids and tamoxifen Overall tamoxifenshowed cytotoxic activity against CHO and MDA-MB-231cells with IC

50values of 8120583gmL and 12 120583gmL respec-

tively compared to MDA-MB-468 cell with IC50

values of27 120583gmL

Morphological changes of cells were observed underan inverted fluorescence microscope (Nikon ECLIPSE Ti-S

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Mersing Muar Skudai Batu Pahat Pulai

Flav

onoi

d co

nten

ts (m

gkg

)

Leaf extracts

KaempferolNaringenin

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

Figure 1 Distribution of kaempferol and naringenin contents inleaf extracts from five different locations Each result is the meanof 3 replicates Error bars represent standard deviations (STDEV)Results that are significantly different lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast119875 lt 0001 are marked with an asterisk

Table 1 Comparison of IC50 values between J gendarussa leafextracts flavonoids and tamoxifen in breast cancer cell lines

IC50 values (120583gmL)MDA-MB-231 MDA-MB-468 CHO

Leaf extractMersing 40 23 28Muar 275 160 108Skudai 61 259 88Batu Pahat 538 398 190Pulai 250 299 305

CompoundsKaempferol 34 23 14Naringenin 238 70 21Mixture of kaempferoland naringenin 43 44 NT

Tamoxifen 12 27 8NT not tested

Shinagawa-ku Tokyo Japan) (100x magnification) after 72hours of treatmentThemethanolic leaf extracts from variouslocations were used to treat MDA-MB-231 cancer cell linesand revealed morphology changes (Figures 2(b) 2(c) 2(d)2(e) and 2(f)) compared to nontreated cells (Figure 2(a))

Morphological changes were revealed after methanolicleaf extract treatment ofMDA-MB-468 cancer cell lines (Fig-ures 3(b) 3(c) 3(d) 3(e) and 3(f)) compared to nontreatedcells (Figure 3(a))

The morphology changes of MDA-MB-231 (Figures 4(b)4(c) and 4(d)) and MDA-MB-468 (Figures 4(f) 4(g) and


(a) (b) (c)

(d) (e) (f)

Figure 2 Morphology changes of MDA-MB-231 cells when treated with leaf extracts (a) MDA-MB-231 cells without any treatment (b) leafextract from Mersing (IC

50 40 120583gmL) (c) leaf extract from Muar (IC

50 275 120583gmL) (d) leaf extract from Skudai (IC

50 61 120583gmL) (e) leaf

extract from Batu Pahat (IC50 538 120583gmL) and (f) leaf extract from Pulai (IC

50 250 120583gmL) Scale bars 100 120583M

4(h)) cancer cell lines when treated with kaempferol narin-genin and a mixture of kaempferol and naringenin comparedto nontreated MDA-MB-231 and MDA-MB-468 cancer celllines (Figures 4(a) and 4(e)) respectively

4 Discussion

Phytochemical analysis of kaempferol and naringenin inleaf extracts from five locations was evaluated and shownin Figure 1 The highest concentrations of kaempferol andnaringenin were found in leaf extracts from Mersing with159180mgkg and 44435mgkg respectively Positive corre-lations were observed between kaempferol and naringenin inall leaf extracts when analysed using the Pearson method Inaddition there was a significant difference in the kaempferoland naringenin distribution contents of leaf extracts fromfivedifferent locations

The cytotoxicity profile of J gendarussa leaf extractsfrom five different locations and flavonoids (kaempferolnaringenin and a mixture of kaempferol and naringenin)against MDA-MD-231 MDA-MB-468 and CHO cells areshown in Table 1 The inhibitory effects of all leaf extractsagainst breast cancer cell lines were decreased in a dosedependent manner and these trends are consistent withprevious studies [10 42 45 46] The IC

50values of the leaf

extract from Mersing (40 120583gmL) showed weak cytotoxicityfollowed by leaf extracts from Skudai (61120583gmL) Batu Pahat

(250 120583gmL) Muar (275120583gmL) and Pulai (275120583gmL)against MDA-MB-231 breast cancer cells IC

50values of the

leaf extract fromMersing (23 120583gmL) showed weak cytotoxi-city followed by leaf extracts fromMuar (160120583gmL) Skudai(259 120583gmL) Batu Pahat (299 120583gmL) and Pulai (398 120583gmL)against MDA-MB-468 cell lines The percent cell viabilityof leaf extracts and flavonoids was compared to the control(untreated cell) The results demonstrate that there was asignificant difference in IC

50values of each leaf extract against

MDA-MB-231 and MDA-MB-468 cell lines (Tables 2 and 3)Because both flavonoids were present in high concentrationsin leaf extracts it is suggested that cytotoxic effects weremainly due to the presence of these flavonoids in elucidatingtumour suppressive effects

Table 1 also shows the ability of kaempferol naringeninand a mixture of kaempferol and naringenin to inhibit theproliferation of breast cancer cell lines in this study How-ever kaempferol showed weak cytotoxicity with IC

50values

of approximately 34 120583gmL (MDA-MB-231) and 23120583gmL(MDA-MB-468) This was followed by naringenin withIC50values of approximately 238 120583gmL (MDA-MB-231) and

70 120583gmL (MDA-MB-468) The mixture of flavonoids alsoshowed weak cytotoxicity with IC

50values of approximately

43 120583gmL (MDA-MB-231) and 44120583gmL (MDA-MB-468) Itis proposed that kaempferol associated highest cytotoxicityagainst breast cancer cell lines compared to naringenin anda mixture of kaempferol and naringenin Table 4 shows that


Table2Percentage

viabilityof

MDA-

MB-231cellsin

leafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin

g6025plusmn006lowastlowastlowast

5435plusmn005lowastlowastlowast


3391plusmn

007lowastlowastlowast




2985plusmn008lowastlowast

Leafextract

from

Muar

7926plusmn007lowast

7289plusmn007lowast

7135plusmn008lowast

7034plusmn007lowast

6349plusmn009lowast




Leafextract

from

Skud

ai6741plusmn

003lowastlowast




4961plusmn





Leafextract

from

Batu

Pahat

8267plusmn007lowast


7720plusmn002lowast






Leafextract

from

Pulai

7461plusmn

008lowastlowast


6924plusmn009lowast


6544plusmn010lowast

6058plusmn007lowast


1671plusmn


Values

arem

eanplusmnST

DEV

for3

replicateslowast119875lt005lowastlowast119875lt001andlowastlowastlowast119875lt0001com

paredto

control(un

treated

cell)


Table3Percentage

viabilityof

MDA-

MB-46

8cells

inleafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin

g9263plusmn004

7653plusmn002lowast

1421plusmn







Leafextract

from

Muar



6920plusmn009lowast


637plusmn001lowast




Leafextract

from

Skud

ai9803plusmn0082

1016

7plusmn0021

9787plusmn0016






Leafextract

from

Batu

Pahat

9626plusmn002

9206plusmn003lowast

9123plusmn009

890plusmn004lowast

10246plusmn006

6461plusmn

008lowast



Leafextract

from

Pulai

9296plusmn003

8868plusmn003lowast







Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table4Percentage

viabilityof

MDA-

MB-231and

MDA-

MB-46

8cells

inkaem

pferoln

aringenin

andam

ixture

ofkaem

pferolandnarin

genin

Cells

Con

centratio

n(120583gmL)

391

781

1563

3125

625

125

250

500

MDA-

MB-231

Kaem

pferol

8216plusmn006


6148plusmn15

9lowastlowast


3645plusmn004lowast

2745plusmn006

4352plusmn002lowast

7737plusmn330lowast

Naringenin

8553plusmn006lowast

10023plusmn006

8713plusmn007


9379plusmn007

8397plusmn004lowast

4791plusmn

004lowastlowast


Mixture

ofkaem

pferolandnarin

genin

7242plusmn011



5610plusmn012lowast





MDA-

MB-46

8Ka

empferol

9600plusmn007








Naringenin

8562plusmn203

9426plusmn230

11771plusmn

007lowast

11218plusmn326

5808plusmn15

1lowast1354plusmn002lowastlowastlowast



Mixture

ofkaem

pferolandnarin

genin

9202plusmn011


8399plusmn098






Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


(a) (b) (c)

(d) (e) (f)

Figure 3 Morphological changes of MDA-MB-468 cells when treated with leaf extracts (a) MDA-MB-468 cells without any treatment (b)leaf extract from Mersing (IC


50 160120583gmL) (d) leaf extract from Skudai (IC

50 259 120583gmL) (e)

leaf extract from Batu Pahat (IC50 398 120583gmL) and (f) leaf extract from Pulai (IC

50 299 120583gmL) Scale bars 100120583M

there was a significant difference between the control withMDA-MB-231 andMDA-MB-468 treated cells for IC

50values

of flavonoids except for kaempferol against MDA-MD-231The leaf extracts and flavonoids also showed low cytotoxicitytoward CHO cells (Table 1)This indicates a lack of selectivityin the cytotoxicity between cancer and normal cells by the leafextracts and flavonoids [47]

However the current study also has contradictory resultsIt is shown in Figures 1 2 and 3 that treated cells showedmoreprominent growth inhibition and shrinkage of the cells whencompared to untreated cells that remained confluent Manyfactors may have influenced these contradictory resultsThe plant source environmental and geographic conditionscell lines and seeding number used in this study werecompletely different from those used in publishedworks [48ndash50] Thus the results presented in this study were not totallyin agreement with published [40 43 44] statements of IC

50

values ranging toward crude extracts Moreover differentplant extracts exhibited different effects on the proliferationof cells according to properties of the compounds [48] Thiswas because selectivity could be due to the sensitivities of celllines against the active compounds in crude extracts that havea specific response [51 52] Overall J gendarussa leaf extractsand flavonoids were considered to hold promising anticancereffects on MDA-MB-231 and MDA-MB-468 cells

The results of J gendarussa leaf extract from Mersingshowed less of an effect against MDA-MB-231 compared

to MDA-MB-468 (Table 1) This suggests that the effectsof active compounds particularly flavonoids on MDA-MB-231 are less cytotoxic compared to those on MDA-MB-468 cell lines MDA-MB-231 is an oestrogen receptor (ER-negative) cell line that containsmore than one cell populationand is highly aggressive invasive and poorly differentiatedfrom human breast cancer cell lines [53 54] MDA-MB-468 cells were most resistant to hyperacetylation and DNAdegradation by drug treatmentsThis suggests that theMDA-MB-468 cell line has a phenotypic difference from and isless invasive than MDA-MB-231 [4] In a previous study Tcrispa and M calabura methanolic leaf extracts producedIC50

values of approximately 525 120583gmL and more than100 120583gmL respectively [10 42] However J gendarussa leafextract from Mersing showed an IC

50value of 40 120583gmL

exhibiting higher toxicity compared to other leaf extracts Itis suggested that J gendarussa leaf extract from Mersing hascytotoxicity potential against MDA-MB-231 cells comparedto other plant leaf extracts

Based on the collected data kaempferol showed thehighest cytotoxicity against MDA-MB-468 followed byMDA-MB-231 and naringenin These results are consistentwith other studies showing weak inhibition of naringeninby other flavonoids [55] A previous study reported thatflavonoidswith hydroxyl substituents at the 41015840 and 7 positionswere invariably oestrogenic and an additional hydroxylgroup at the 5th position increased estrogenic activity [56]


(a) (b) (c)

(d) (e) (f)

(g) (h)

Figure 4 Morphology of MDA-MB231 and MDA-MB-468 cells when treated with kaempferol naringenin and a mixture of kaempferoland naringenin (a) MDA-MB-231 cells without any treatment control (b) MDA-MB-231 cells treated with kaempferol (IC

50 34 120583gmL) (c)

MDA-MB-231 cells treated with naringenin (IC50 238 120583gmL) (d) MDA-MB-231 cells treated with a mixture of kaempferol and naringenin

(IC50 43 120583gmL) (e)MDA-MB-468 cells without any treatment control (f)MDA-MB-468 cells treatedwith kaempferol (IC

50 23 120583gmL) (g)

MDA-MB-468 cells treated with naringenin (IC50 70 120583gmL) (h) MDA-MB-468 cells treated with a mixture of kaempferol and naringenin

(IC50 44120583gmL) Scale bars 100 120583M

The present study supports this claim [56] Previous workalso demonstrated that naringenin showed a stronger oestro-genicitywhen tested onBT-474 humanbreast cancer cell lines[57] It is plausible to suggest that both flavonoids contributestrong oestrogenic potency to the inhibition of oestrogen-independent breast cancer cells MDA-MB-231 and MDA-MB-468

Table 1 also shows the cytotoxicity of J gendarussaleaf extracts flavonoids and tamoxifen on a normal cellline (CHO) CHO cells were a positive control used forcomparison with the cytotoxicity activity on MDA-MB-231

and MDA-MB-468 breast cancer cell lines Comparisons ofJ gendarussa leaf extracts flavonoids and tamoxifen wereperformed in terms of IC

50values between breast cancer

and normal cell lines Tamoxifen was demonstrated to becytotoxic to CHO cell lines (IC

50lt 20120583gmL) in this study

Although the IC50values of leaf extracts and flavonoids were

not as low as tamoxifen they had low toxicity against CHOcells Due to its high toxicity in CHO cells the continuoususe of tamoxifen can cause adverse side effects [58] Ifthese results also occur in vivo these leaf extracts would beconsidered safe for human consumption and could be used


for further toxicity and clinical studies Hence the use of leafextracts and flavonoids as anticancer agents in combinationwith other therapeutic drugs may reduce the adverse effectsof drugs Therefore more comprehensive studies involvinganimal and clinical investigations are required

5 Conclusion

In conclusion J gendarussa leaf extract from Mersing andkaempferol were considered cytotoxic against MDA-MB-231 and MDA-MB-468 compared to other leaf extracts andnaringenin Leaf extract fromMersing showed high contentsof kaempferol and naringenin compared to other leaf extractswhen quantified usingGC-FIDOur results suggest that thereis a correlation between the presence of kaempferol in the leafextract from Mersing with the level of cytotoxicity againstboth breast cancer cell lines These data will be beneficialto other researchers and validate the potential use of Jgendarussa leaves as novel anticancer agents

Conflict of Interests

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

Acknowledgments

The authors thank Universiti Teknologi Malaysia (UTM)and the Ministry of Education Malaysia (MOE) (MyPhD)for financial support and Animal Tissue Culture Faculty ofBiosciences and Medical Engineering for lab facilities

References

[1] T Kummalue P O-charoenrat W Jiratchariyakul et alldquoAntiproliferative effect of Erycibe elliptilimba on human breastcancer cell linesrdquo Journal of Ethnopharmacology vol 110 no 3pp 439ndash443 2007

[2] N C Registry Malaysia Cancer StatisticsmdashData and Figure2007 Malaysia Ministry of Health 2007

[3] E L Jones L R Prosnitz MW Dewhirst et al ldquoThermochem-oradiotherapy improves oxygenation in locally advanced breastcancerrdquo Clinical Cancer Research vol 10 no 13 pp 4287ndash42932004

[4] C R Tate L V Rhodes H C Segar et al ldquoTargeting triple-neg-ative breast cancer cells with the histone deacetylase inhibitorpanobinostatrdquo Breast Cancer Research vol 14 no 79 pp 1ndash152012

[5] R Cailleau BMackay R K Young andW J Reeves Jr ldquoTissueculture studies on pleural effusions from breast carcinomapatientsrdquo Cancer Research vol 34 no 4 pp 801ndash809 1974

[6] J B Engel A V Schally G Halmos B Baker A Nagyand G Keller ldquoTargeted cytotoxic bombesin analog AN-215effectively inhibits experimental human breast cancers with alow induction of multi-drug resistance proteinsrdquo Endocrine-Related Cancer vol 12 no 4 pp 999ndash1009 2005

[7] R Cailleau M Olive and Q V J Cruciger ldquoLong-term humanbreast carcinoma cell lines of metastatic origin preliminarycharacterizationrdquo In Vitro vol 14 no 11 pp 911ndash915 1978

[8] A C Figueroa E A Soria J J Cantero et al ldquoCytotoxic activityofThelespermamegapotamicum organic fractions againstMCF-7 human breast cancer cell linerdquo Journal of CancerTherapy vol3 pp 103ndash109 2012

[9] B Rajkapoor B Jayakar and N Murugesh ldquoAntitumor activityof Indigofera aspalathoides on Ehrlich ascites carcinoma inmicerdquo Indian Journal of Pharmacology vol 36 no 1 pp 38ndash402004

[10] Z A Zakaria A M Mohamed N S M Jamil et al ldquoIn vitroantiproliferative and antioxidant activities of the extracts ofMuntingia calabura leavesrdquo The American Journal of ChineseMedicine vol 39 no 1 pp 183ndash200 2011

[11] T D Thomas and H Yoichiro ldquoIn vitro propagation for theconservation of a raremedicinal plant Justicia gendarussaBurmf by nodal explants and shoot regeneration from callusrdquo ActaPhysiologiae Plantarum vol 32 no 5 pp 943ndash950 2010

[12] B JanarthanamandE Sumathi ldquoIn vitro regeneration of Justiciagendarussa Burm frdquo Libyan Agriculture Research Center JournalInternational vol 1 no 5 pp 284ndash287 2010

[13] E W B Prajogo F Ifadotunnikmah A P Febriyanti et alldquoEffect of Justicia gendarussa Burmf leaves water fraction onmale rabbit liver and renal fuction (Sub acute toxicity testof Justicia gendarussa Burmf leaves water fraction as malecontraceptive agent)rdquo Veterinaria Medika vol 1 no 3 pp 79ndash82 2008

[14] S S Patel andM N Zaveri ldquoCytotoxic activity to find bioactivecompound from Justicia gendarussa using brine shrimp lethal-ity assayrdquoAsian Journal of TraditionalMedicines vol 7 no 3 pp102ndash108 2012

[15] K Periyanayagam B Umamaheswari L Suseela M Padminiand M Ismail ldquoEvaluation of antiangiogenic effect of theleaves of Justicia gendarussa (Burm f) (Acanthaceae) by chrioallontoic membrane methodrdquo American Journal of InfectiousDiseases vol 5 no 3 pp 187ndash189 2009

[16] K L Krishna T A Mehta and J A Patel ldquoIn-vitro hepato-protective activity of Justicia gendarussa stem on isolated rathepatocytesrdquo Pharmacologyonline vol 2 pp 9ndash13 2010

[17] KK Sharma R Saikia J Kotoky J CKalita andRDevi ldquoAnti-fungal activity of Solanum melongena L Lawsonia inermis Land Justicia gendarussa B against dermatophytesrdquo InternationalJournal of PharmTech Research vol 3 no 3 pp 1635ndash1640 2011

[18] M R Uddin S Sinha M A Hossain M A Kaisar M K Hos-sain andM A Rashid ldquoChemical and biological investigationsof Justicia gendarussa (Burm F)rdquo Dhaka University Journal ofPharmaceutical Sciences vol 10 no 1 pp 53ndash57 2011

[19] M F Fazaludeen C Manickam I M A Ashankyty et al ldquoSyn-thesis and characterizations of gold nanoparticles by Justiciagendarussa Burm F leaf extractrdquo Journal of Microbiology andBiotechnology Research vol 2 no 1 pp 23ndash34 2012

[20] M R Saha P C Debnath M A Rahman and M A UIslam ldquoEvaluation of in vitro anthelmintic activities of leafand stem extracts of Justicia gendarussardquo Bangladesh Journal ofPharmacology vol 7 no 1 pp 50ndash53 2012

[21] N Subramanian C Jothimanivannan and K MoorthyldquoAntimicrobial activity and preliminary phytochemicalscreening of Justicia gendarussa (Burm f) against humanpathogensrdquo Asian Journal of Pharmaceutical and ClinicalResearch vol 5 no 3 pp 229ndash233 2012

[22] V S Sudhanandh J K Arjun A K Faisal et al ldquoIn-vitroantibacterial screening of selected folklore Indian medicinalplants with few clinical pathogensrdquo Indian Journal of Pharma-ceutical Education and Research vol 46 no 2 pp 174ndash178 2012


[23] D Kowsalya and S Sankaranarayanan ldquoEfficacies of bacteri-cidal Justicia gendarussa extract inhibiting protein synthesisagainst methicilin resistant Staphylococcus aureusrdquo IOSR Jour-nal of Pharmacy and Biological Sciences vol 4 no 2 pp 32ndash412012

[24] R A Mustafa A A Hamid S Mohamed and F A BakarldquoTotal phenolic compounds flavonoids and radical scavengingactivity of 21 selected tropical plantsrdquo Journal of Food Sciencevol 75 no 1 pp 28ndash35 2010

[25] E B Prajogo D Guliet E Ferreira Queiroz et al ldquoIsolationof male antifertility compound in n-butanol fraction of Justiciagendarussa Burm F leavesrdquo Folia Medica Indonesiana vol 45no 1 pp 28ndash31 2009

[26] A K Chakravarty P P G Dastidar and S C Pakrashi ldquoSimplearomatic amines from Justicia gendarussa 13C NMR spectra ofthe bases and their analoguesrdquo Tetrahedron vol 38 no 12 pp1797ndash1802 1982

[27] WDRatnasooriya S ADeraniyagala andDCDehigaspitiyaldquoAntinociceptive activity and toxicological study of aqueous leafextract of Justicia gendarussa Burm F in ratsrdquo PharmacognosyMagazine vol 3 no 11 pp 145ndash155 2007

[28] Z Ayob N H M Saari and A A Samad ldquoIn vitro propagationand flavonoid contents in local Justicia gendarussa Burm Frdquo inProceedings of the 11th International Annual Symposium on Sus-tainability Science and Management pp 403ndash409 TerengganuMalaysia July 2012

[29] Z Ayob A Abd Samad and S P Mohd Bohari ldquoCytotoxicityactivities in local Justicia gendarussa crude extracts againsthuman cancer cell linesrdquo Jurnal Teknologi (Sciences and Engi-neering) vol 64 no 2 pp 45ndash52 2013

[30] Y Zhang A Y ChenM Li C Chen andQ Yao ldquoGinkgo bilobaextract kaempferol inhibits cell proliferation and induces apop-tosis in pancreatic cancer cellsrdquo Journal of Surgical Research vol148 no 1 pp 17ndash23 2008

[31] M Cavia-Saiz M D Busto M C Pilar-Izquierdo N OrtegaM Perez-Mateos and PMuniz ldquoAntioxidant properties radicalscavenging activity and biomolecule protection capacity offlavonoid naringenin and its glycoside naringin a comparativestudyrdquo Journal of the Science of Food and Agriculture vol 90 no7 pp 1238ndash1244 2010

[32] K-T Kim E-J Yeo S-H Moon et al ldquoInhibitory effects ofnaringenin kaempherol and apigenin on cholesterol biosyn-thesis in HepG2 and MCF-7 cellsrdquo Food Science and Biotech-nology vol 17 no 6 pp 1361ndash1364 2008

[33] Y-C Tu T-W Lian J-H Yen Z-T Chen and M-J WuldquoAntiatherogenic effects of kaempferol and rhamnocitrinrdquo Jour-nal of Agricultural and Food Chemistry vol 55 no 24 pp 9969ndash9976 2007

[34] D Susanti H M Sirat F Ahmad R M Ali N Aimi andM Kitajima ldquoAntioxidant and cytotoxic flavonoids from theflowers of Melastoma malabathricum Lrdquo Food Chemistry vol103 no 3 pp 710ndash716 2007

[35] J-H Park J-W Lee H-D Paik et al ldquoCytotoxic effects of 7-O-butyl naringenin on human breast cancerMCF-7 cellsrdquo FoodScience and Biotechnology vol 19 no 3 pp 717ndash724 2010

[36] S-I Kanno A Tomizawa T Hiura et al ldquoInhibitory effects ofnaringenin on tumor growth in human cancer cell lines andsarcoma S-180-implanted micerdquo Biological and PharmaceuticalBulletin vol 28 no 3 pp 527ndash530 2005

[37] A Kamuhabwa C Nshimo and P De Witte ldquoCytotoxicity ofsome medicinal plant extracts used in Tanzanian traditional

medicinerdquo Journal of Ethnopharmacology vol 70 no 2 pp 143ndash149 2000

[38] N Sarju A Samad M Ghani and F Ahmad ldquoDetection andquantification of Naringenin and kaempferol in Melastomadecemfidum extracts by GC-FID and GC-MSrdquo Acta Chromato-graphica vol 24 no 2 pp 221ndash228 2012

[39] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[40] R Ahmad A M Ali D A Israf N H Ismail K Shaari and NH Lajis ldquoAntioxidant radical-scavenging anti-inflammatorycytotoxic and antibacterial activities of methanolic extracts ofsome Hedyotis speciesrdquo Life Sciences vol 76 no 17 pp 1953ndash1964 2005

[41] J Pallant SPSS Survival Manual McGraw-Hill New York NYUSA 2007

[42] M J Ibahim I W M Z Wan-Nor A H H Narimah A ZNurul S S A R Siti-Nur and G A Froemming ldquoAnti-proli-perative and antioxidant effects of Tinospora crispa (Batawali)rdquoBiomedical Research vol 22 no 1 pp 57ndash62 2011

[43] R I Geran H M Greenberg M McDonald et al ldquoPro-tocols for screening chemical agents and natural productsagainst animal tumors and other biological systemsrdquo CancerChemotherapy Reports vol 33 pp 1ndash17 1972

[44] SMMohamedAMAliM Rahmani CWiart J S Dhaliwaland K Yusoff ldquoApoptotic and necrotic cell deathmanifestationsin leukemic cells treated with methylgerambullin a sulphonefrom Glycosmis calcicolardquo Journal of Biochemistry MolecularBiology and Biophysics vol 4 no 4 pp 253ndash261 2000

[45] H Z Chong A Rahmat S K Yeap et al ldquoIn vitro cytotoxicityof Strobilanthes crispus ethanol extract on hormone dependenthuman breast adenocarcinoma MCF-7 cellrdquo BMC Complemen-tary and Alternative Medicine vol 12 article 35 2012

[46] A Subarnas A Diantini R Abdulah et al ldquoAntiproliferativeactivity of primates-consumed plants against MCF-7 humanbreast cancer cell linesrdquo E3 Journal of Medical Research vol 1no 4 pp 38ndash43 2012

[47] N Armania L S Yazan I S Ismail et al ldquoDillenia suffruticosaextract inhibits proliferation of human breast cancer cell lines(MCF-7 and MDA-MB-231) via Induction of G2M arrest andapoptosisrdquoMolecules vol 18 no 11 pp 13320ndash13339 2013

[48] R K Al-Barazanjy K Dizaye and A AL-Asadye ldquoCytotoxicand cytogenetic effects of Salvia officinalis on different tumorcell linesrdquoMiddle East Journal of International Medicine vol 6no 4 pp 15ndash25 2013

[49] A Alsemari F Alkhodairy A Aldakan et al ldquoThe selectivecytotoxic anti-cancer properties and proteomic analysis ofTrigonella Foenum-Graecumrdquo BMC Complementary and Alter-native Medicine vol 14 article 114 2014

[50] J Han T P Talorete P Yamada and H Isoda ldquoAnti-prolifera-tive and apoptotic effects of oleuropein and hydroxytyrosol onhuman breast cancer MCF-7 cellsrdquo Cytotechnology vol 59 no1 pp 45ndash53 2009

[51] C Kirana I R Record G H McIntosh and G P JonesldquoScreening for antitumor activity of 11 species of Indonesianzingiberaceae using human MCF-7 and HT-29 cancer cellsrdquoPharmaceutical Biology vol 41 no 4 pp 271ndash276 2003

[52] C G Joshi M Gopal and S M Byregowda ldquoCytotoxic activityof Tragia involucrata Linn extractsrdquo American-Eurasian Jour-nal of Toxicological Sciences vol 3 no 2 pp 67ndash69 2011


[53] H Liu C Zang M H Fenner K Possinger and E ElstnerldquoPPAR120574 ligands andATRA inhibit the invasion of human breastcancer cells in vitrordquo Breast Cancer Research and Treatment vol79 no 1 pp 63ndash74 2003

[54] K B Yin ldquoThe mesenchymal-like phenotype of the MDA-MB-231 cell line breast cancermdashfocusing tumormicroenvironmentstem cells and metastasisrdquo in Breast CancermdashFocusing TumorMicroenvironment Stem cells andMetastasis PM Gunduz Edvol 18 pp 385ndash402 InTech 2011

[55] J C Le Bail F Varnat J C Nicolas and G HabriouxldquoEstrogenic and antiproliferative activities on MCF-7 humanbreast cancer cells by flavonoidsrdquo Cancer Letters vol 130 no1-2 pp 209ndash216 1998

[56] F V So N Guthrie A F Chambers et al ldquoInhibition ofproliferation of estrogen receptor-positiveMCF-7 humanbreastcancer cells by flavonoids in the presence and absence of excessestrogenrdquo Cancer Letters vol 112 pp 127ndash133 1997

[57] R S Rosenberg Zand D J A Jenkins and E P DiamandisldquoSteroid hormone activity of flavonoids and related com-poundsrdquo Breast Cancer Research and Treatment vol 62 no 1pp 35ndash49 2000

[58] M Lazzeroni D Serrano B K Dunn et al ldquoOral low doseand topical tamoxifen for breast cancer prevention modernapproaches for an old drugrdquo Breast Cancer Research vol 14article 214 2012


Lanka India and Malaysia [11] The roots and leaf extractsof J gendarussa have been demonstrated to treat chronicrheumatism inflammation bronchitis headache arthri-tis vaginal discharges dyspepsia eye disease and fever[12]

Previous reports demonstrated that J gendarussa leafextracts have been used traditionally as a male contraceptiveagent by several ethnic groups in the central part of PapuaIndonesia This extract is able to inhibit mouse spermatozoapenetration of mice ovum [13] J gendarussa methanolicleaves and root extracts showed cytotoxic activity againstbrine shrimp in the brine shrimp lethality assay with IC

50

values of 4871 120583gmL and 9325 120583gmL respectively [14] Inaddition J gendarussa leaves and stem extracts were reportedto have anticancer antioxidant antibacterial antifungalantiangiogenic anthelmintic and hepatoprotective activities[15ndash23]

Phytochemical studies on leaves from J gendarussarevealed the presence of flavonoids alkaloids triterpenoidalsaponins amino acids aromatic amines stigmasterol andlupeol [18 24ndash27] Our previous study on green callus andin vitro leaf extracts of J gendarussa detected two flavonoidsthat is kaempferol and naringenin using GC-FID [28] Bothflavonoids were also detected in themethanolic leaf extract ofJ gendarussa using the same method [29] Bioactivity studieson both flavonoids found that it exhibited strong antioxidantand inhibitory effects on cholesterol in HepG2 cancer cells[30ndash32] Kaempferol also inhibited pancreatic cancer cell(MIAPaCa-2 and Panc-1) proliferation induced cancer cellapoptosis and prevented arteriosclerosis [30 33] Naringenindemonstrated cytotoxic effects against breast cancer cells(MCF-7) and suppressed apoptosis in mouse leukaemia P388cells [34ndash36] Our previous study on both flavonoids showedstrong cytotoxic activity against colonic (HT-29) cervical(HeLa) and pancreatic (BxPC-3) cells [29]

To the best of our knowledge this is the first study of theeffects of J gendarussa leaf extracts against human breast can-cer cell lines (MDA-MB-231 and MDA-MB-468) This studywas performed to screen the cytotoxic activities ofmethanolicleaf extracts from five different locations (Mersing MuarSkudai Batu Pahat and Pulai) in Johor and two flavonoids(naringenin and kaempferol) against breast cancer cell linesThe quantification of kaempferol and naringenin content inleaf extracts of J gendarussa using GC-FID was also carriedout

2 Methods and Materials

21 Plant Materials J gendarussa plants were collected fromfive different locations in Johor (Mersing Muar Skudai BatuPahat and Pulai) and maintained in a greenhouse at theFaculty of Biosciences and Medical Engineering UniversitiTeknologi Malaysia (UTM) The J gendarussa plant wasidentified byDr RichardChungChengKong senior researchofficer of the Forest Research Institute of Malaysia (FRIM)The voucher specimen (PID-100214-06) was deposited atHerbarium Management Branch Flora Biodiversity Pro-gram Forest Biodiversity Division FRIM Kepong SelangorMalaysia

22 General Chemicals Commercial standards (kaempferoland naringenin) were purchased from Sigma-Aldrich (Sub-ang Jaya Selangor Malaysia) Tamoxifen was used as apositive control in the MTT assay All samples were dilutedwith 01 of dimethylsulfoxide (DMSO) which has no effecton cell viability [37]

23 Preparation of Extracts The J gendarussa leaves were air-dried for 4 weeks The dried leaves were ground into smallparticles and approximately 50 g of small particles was soakedinto 1000mLofmethanol at room temperature for 72 hours ina ratio of 1 20 (wv) [10] The mixtures were filtered throughsterile cotton and filtered again using Whatman number 1filter paper to obtain methanolic supernatants The filteredmethanolic extract was evaporated at 40∘C under reducedpressure by using an EYELA N-1000 rotary evaporator(Bohemia NY USA)The dried crude extract was kept at 4∘Cprior to use

24 Quantification of Flavonoids in Leaf Extracts GC-FIDand quantitative analysis were performed according to previ-ously published method [38] GC-FID (HP-6890N AgilentUSA) equipped with a HP-5 fused silica capillary column(300m times 032mm ID times 025120583m) was used The temperatureprogrammed was 100∘C held for 1 minute and then rampedto 275∘C at 10∘Cmin and held for 17 minutes at 275∘C Theinjection temperature was 275∘C The flow rate of the carriergas (helium) was 1mLmin A split ratio of 50 1 was used Aquantity of 5120583L of leaf extract and standardswas injectedThechromatographic data were recorded and processed usingAgilent Cerity QA-QC software

25 Cell Culture MDA-MB-231 (basal-B mammary carci-noma) and MDA-MB-468 (basal-A mammary carcinoma)breast cancer cell lines and CHO (Chinese hamster ovary)normal cell line were obtained from American Type CultureCollection (ATCC) and as a generous gift from Dr Sale-hhuddin Hamdan (Animal Cell Culture Laboratory Facultyof Biosciences and Medical Engineering UTM) MDA-MB-231 and MDA-MB-468 breast cancer cell lines were culturedin Dulbeccorsquos modified Eaglersquos medium (DMEM) whileCHO normal cells were cultured in Roswell Park MemorialInstitute 1640 (RPMI-1640) medium supplemented with 10vv foetal bovine serum (FBS) 100UmL of penicillin and100 120583gmL of streptomycin as a complete growth mediumCells were maintained in 25 cm2 flasks and incubatedin a humidified incubator (CO

2Water-Jacketed Incubator

NuAire Fernbrook Lane Plymouth USA) at 37∘C with 5CO2 All materials were obtained from Gibco (Gibco Bio-

Diagnostics Petaling Jaya Selangor Malaysia)

26 MTTAssay Cytotoxicity testing was performed using 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT Sigma) according to the method reported in previousstudies [29 39] In this assay cells were harvested afterreaching 80 confluence Before starting theMTT assay cellswere optimised at different seeding densities ranging from20 times 103 cellmL to 10 times 106 cellmL in light to determine





3 Results



50


50values where IC









0

200

400

600

800

1000

1200

1400

1600

1800

2000


Flav

onoi

d co

nten

ts (m

gkg

)

Leaf extracts


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast











(a) (b) (c)

(d) (e) (f)




50 61 120583gmL) (e) leaf


50 250 120583gmL) Scale bars 100 120583M


4 Discussion






50values of the





50values






Table2Percentage

viabilityof

MDA-

MB-231cellsin

leafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin




3391plusmn






Leafextract

from

Muar

7926plusmn007lowast

7289plusmn007lowast

7135plusmn008lowast

7034plusmn007lowast

6349plusmn009lowast




Leafextract

from

Skud

ai6741plusmn

003lowastlowast




4961plusmn





Leafextract

from

Batu

Pahat

8267plusmn007lowast


7720plusmn002lowast






Leafextract

from

Pulai

7461plusmn

008lowastlowast


6924plusmn009lowast


6544plusmn010lowast

6058plusmn007lowast


1671plusmn


Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table3Percentage

viabilityof

MDA-

MB-46

8cells

inleafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin

g9263plusmn004

7653plusmn002lowast

1421plusmn







Leafextract

from

Muar



6920plusmn009lowast


637plusmn001lowast




Leafextract

from

Skud

ai9803plusmn0082

1016

7plusmn0021

9787plusmn0016






Leafextract

from

Batu

Pahat

9626plusmn002

9206plusmn003lowast

9123plusmn009

890plusmn004lowast

10246plusmn006

6461plusmn

008lowast



Leafextract

from

Pulai

9296plusmn003

8868plusmn003lowast







Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table4Percentage

viabilityof

MDA-

MB-231and

MDA-

MB-46

8cells

inkaem

pferoln

aringenin

andam

ixture

ofkaem

pferolandnarin

genin

Cells

Con

centratio

n(120583gmL)

391

781

1563

3125

625

125

250

500

MDA-

MB-231

Kaem

pferol

8216plusmn006


6148plusmn15

9lowastlowast


3645plusmn004lowast

2745plusmn006

4352plusmn002lowast

7737plusmn330lowast

Naringenin

8553plusmn006lowast

10023plusmn006

8713plusmn007


9379plusmn007

8397plusmn004lowast

4791plusmn

004lowastlowast


Mixture

ofkaem

pferolandnarin

genin

7242plusmn011



5610plusmn012lowast





MDA-

MB-46

8Ka

empferol

9600plusmn007








Naringenin

8562plusmn203

9426plusmn230

11771plusmn

007lowast

11218plusmn326

5808plusmn15




Mixture

ofkaem

pferolandnarin

genin

9202plusmn011


8399plusmn098






Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


(a) (b) (c)

(d) (e) (f)




50 259 120583gmL) (e)


50 299 120583gmL) Scale bars 100120583M


50values



50









(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References


































































3 Results



50


50values where IC









0

200

400

600

800

1000

1200

1400

1600

1800

2000


Flav

onoi

d co

nten

ts (m

gkg

)

Leaf extracts


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast











(a) (b) (c)

(d) (e) (f)




50 61 120583gmL) (e) leaf


50 250 120583gmL) Scale bars 100 120583M


4 Discussion






50values of the





50values






Table2Percentage

viabilityof

MDA-

MB-231cellsin

leafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin




3391plusmn






Leafextract

from

Muar

7926plusmn007lowast

7289plusmn007lowast

7135plusmn008lowast

7034plusmn007lowast

6349plusmn009lowast




Leafextract

from

Skud

ai6741plusmn

003lowastlowast




4961plusmn





Leafextract

from

Batu

Pahat

8267plusmn007lowast


7720plusmn002lowast






Leafextract

from

Pulai

7461plusmn

008lowastlowast


6924plusmn009lowast


6544plusmn010lowast

6058plusmn007lowast


1671plusmn


Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table3Percentage

viabilityof

MDA-

MB-46

8cells

inleafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin

g9263plusmn004

7653plusmn002lowast

1421plusmn







Leafextract

from

Muar



6920plusmn009lowast


637plusmn001lowast




Leafextract

from

Skud

ai9803plusmn0082

1016

7plusmn0021

9787plusmn0016






Leafextract

from

Batu

Pahat

9626plusmn002

9206plusmn003lowast

9123plusmn009

890plusmn004lowast

10246plusmn006

6461plusmn

008lowast



Leafextract

from

Pulai

9296plusmn003

8868plusmn003lowast







Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table4Percentage

viabilityof

MDA-

MB-231and

MDA-

MB-46

8cells

inkaem

pferoln

aringenin

andam

ixture

ofkaem

pferolandnarin

genin

Cells

Con

centratio

n(120583gmL)

391

781

1563

3125

625

125

250

500

MDA-

MB-231

Kaem

pferol

8216plusmn006


6148plusmn15

9lowastlowast


3645plusmn004lowast

2745plusmn006

4352plusmn002lowast

7737plusmn330lowast

Naringenin

8553plusmn006lowast

10023plusmn006

8713plusmn007


9379plusmn007

8397plusmn004lowast

4791plusmn

004lowastlowast


Mixture

ofkaem

pferolandnarin

genin

7242plusmn011



5610plusmn012lowast





MDA-

MB-46

8Ka

empferol

9600plusmn007








Naringenin

8562plusmn203

9426plusmn230

11771plusmn

007lowast

11218plusmn326

5808plusmn15




Mixture

ofkaem

pferolandnarin

genin

9202plusmn011


8399plusmn098






Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


(a) (b) (c)

(d) (e) (f)




50 259 120583gmL) (e)


50 299 120583gmL) Scale bars 100120583M


50values



50









(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References































































(a) (b) (c)

(d) (e) (f)




50 61 120583gmL) (e) leaf


50 250 120583gmL) Scale bars 100 120583M


4 Discussion






50values of the





50values






Table2Percentage

viabilityof

MDA-

MB-231cellsin

leafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin




3391plusmn






Leafextract

from

Muar

7926plusmn007lowast

7289plusmn007lowast

7135plusmn008lowast

7034plusmn007lowast

6349plusmn009lowast




Leafextract

from

Skud

ai6741plusmn

003lowastlowast




4961plusmn





Leafextract

from

Batu

Pahat

8267plusmn007lowast


7720plusmn002lowast






Leafextract

from

Pulai

7461plusmn

008lowastlowast


6924plusmn009lowast


6544plusmn010lowast

6058plusmn007lowast


1671plusmn


Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table3Percentage

viabilityof

MDA-

MB-46

8cells

inleafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin

g9263plusmn004

7653plusmn002lowast

1421plusmn







Leafextract

from

Muar



6920plusmn009lowast


637plusmn001lowast




Leafextract

from

Skud

ai9803plusmn0082

1016

7plusmn0021

9787plusmn0016






Leafextract

from

Batu

Pahat

9626plusmn002

9206plusmn003lowast

9123plusmn009

890plusmn004lowast

10246plusmn006

6461plusmn

008lowast



Leafextract

from

Pulai

9296plusmn003

8868plusmn003lowast







Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table4Percentage

viabilityof

MDA-

MB-231and

MDA-

MB-46

8cells

inkaem

pferoln

aringenin

andam

ixture

ofkaem

pferolandnarin

genin

Cells

Con

centratio

n(120583gmL)

391

781

1563

3125

625

125

250

500

MDA-

MB-231

Kaem

pferol

8216plusmn006


6148plusmn15

9lowastlowast


3645plusmn004lowast

2745plusmn006

4352plusmn002lowast

7737plusmn330lowast

Naringenin

8553plusmn006lowast

10023plusmn006

8713plusmn007


9379plusmn007

8397plusmn004lowast

4791plusmn

004lowastlowast


Mixture

ofkaem

pferolandnarin

genin

7242plusmn011



5610plusmn012lowast





MDA-

MB-46

8Ka

empferol

9600plusmn007








Naringenin

8562plusmn203

9426plusmn230

11771plusmn

007lowast

11218plusmn326

5808plusmn15




Mixture

ofkaem

pferolandnarin

genin

9202plusmn011


8399plusmn098






Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


(a) (b) (c)

(d) (e) (f)




50 259 120583gmL) (e)


50 299 120583gmL) Scale bars 100120583M


50values



50









(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References































































Table2Percentage

viabilityof

MDA-

MB-231cellsin

leafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin




3391plusmn






Leafextract

from

Muar

7926plusmn007lowast

7289plusmn007lowast

7135plusmn008lowast

7034plusmn007lowast

6349plusmn009lowast




Leafextract

from

Skud

ai6741plusmn

003lowastlowast




4961plusmn





Leafextract

from

Batu

Pahat

8267plusmn007lowast


7720plusmn002lowast






Leafextract

from

Pulai

7461plusmn

008lowastlowast


6924plusmn009lowast


6544plusmn010lowast

6058plusmn007lowast


1671plusmn


Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table3Percentage

viabilityof

MDA-

MB-46

8cells

inleafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin

g9263plusmn004

7653plusmn002lowast

1421plusmn







Leafextract

from

Muar



6920plusmn009lowast


637plusmn001lowast




Leafextract

from

Skud

ai9803plusmn0082

1016

7plusmn0021

9787plusmn0016






Leafextract

from

Batu

Pahat

9626plusmn002

9206plusmn003lowast

9123plusmn009

890plusmn004lowast

10246plusmn006

6461plusmn

008lowast



Leafextract

from

Pulai

9296plusmn003

8868plusmn003lowast







Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table4Percentage

viabilityof

MDA-

MB-231and

MDA-

MB-46

8cells

inkaem

pferoln

aringenin

andam

ixture

ofkaem

pferolandnarin

genin

Cells

Con

centratio

n(120583gmL)

391

781

1563

3125

625

125

250

500

MDA-

MB-231

Kaem

pferol

8216plusmn006


6148plusmn15

9lowastlowast


3645plusmn004lowast

2745plusmn006

4352plusmn002lowast

7737plusmn330lowast

Naringenin

8553plusmn006lowast

10023plusmn006

8713plusmn007


9379plusmn007

8397plusmn004lowast

4791plusmn

004lowastlowast


Mixture

ofkaem

pferolandnarin

genin

7242plusmn011



5610plusmn012lowast





MDA-

MB-46

8Ka

empferol

9600plusmn007








Naringenin

8562plusmn203

9426plusmn230

11771plusmn

007lowast

11218plusmn326

5808plusmn15




Mixture

ofkaem

pferolandnarin

genin

9202plusmn011


8399plusmn098






Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


(a) (b) (c)

(d) (e) (f)




50 259 120583gmL) (e)


50 299 120583gmL) Scale bars 100120583M


50values



50









(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References































































Table3Percentage

viabilityof

MDA-

MB-46

8cells

inleafextractsfro

mfived

ifferentlocations

Con

centratio

n(120583gmL)

781

1563

3125

625

125

250

500

1000

Leafextract

from

Mersin

g9263plusmn004

7653plusmn002lowast

1421plusmn







Leafextract

from

Muar



6920plusmn009lowast


637plusmn001lowast




Leafextract

from

Skud

ai9803plusmn0082

1016

7plusmn0021

9787plusmn0016






Leafextract

from

Batu

Pahat

9626plusmn002

9206plusmn003lowast

9123plusmn009

890plusmn004lowast

10246plusmn006

6461plusmn

008lowast



Leafextract

from

Pulai

9296plusmn003

8868plusmn003lowast







Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


Table4Percentage

viabilityof

MDA-

MB-231and

MDA-

MB-46

8cells

inkaem

pferoln

aringenin

andam

ixture

ofkaem

pferolandnarin

genin

Cells

Con

centratio

n(120583gmL)

391

781

1563

3125

625

125

250

500

MDA-

MB-231

Kaem

pferol

8216plusmn006


6148plusmn15

9lowastlowast


3645plusmn004lowast

2745plusmn006

4352plusmn002lowast

7737plusmn330lowast

Naringenin

8553plusmn006lowast

10023plusmn006

8713plusmn007


9379plusmn007

8397plusmn004lowast

4791plusmn

004lowastlowast


Mixture

ofkaem

pferolandnarin

genin

7242plusmn011



5610plusmn012lowast





MDA-

MB-46

8Ka

empferol

9600plusmn007








Naringenin

8562plusmn203

9426plusmn230

11771plusmn

007lowast

11218plusmn326

5808plusmn15




Mixture

ofkaem

pferolandnarin

genin

9202plusmn011


8399plusmn098






Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


(a) (b) (c)

(d) (e) (f)




50 259 120583gmL) (e)


50 299 120583gmL) Scale bars 100120583M


50values



50









(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References































































Table4Percentage

viabilityof

MDA-

MB-231and

MDA-

MB-46

8cells

inkaem

pferoln

aringenin

andam

ixture

ofkaem

pferolandnarin

genin

Cells

Con

centratio

n(120583gmL)

391

781

1563

3125

625

125

250

500

MDA-

MB-231

Kaem

pferol

8216plusmn006


6148plusmn15

9lowastlowast


3645plusmn004lowast

2745plusmn006

4352plusmn002lowast

7737plusmn330lowast

Naringenin

8553plusmn006lowast

10023plusmn006

8713plusmn007


9379plusmn007

8397plusmn004lowast

4791plusmn

004lowastlowast


Mixture

ofkaem

pferolandnarin

genin

7242plusmn011



5610plusmn012lowast





MDA-

MB-46

8Ka

empferol

9600plusmn007








Naringenin

8562plusmn203

9426plusmn230

11771plusmn

007lowast

11218plusmn326

5808plusmn15




Mixture

ofkaem

pferolandnarin

genin

9202plusmn011


8399plusmn098






Values

arem

eanplusmnST

DEV

for3


paredto

control(un

treated

cell)


(a) (b) (c)

(d) (e) (f)




50 259 120583gmL) (e)


50 299 120583gmL) Scale bars 100120583M


50values



50









(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References































































(a) (b) (c)

(d) (e) (f)




50 259 120583gmL) (e)


50 299 120583gmL) Scale bars 100120583M


50values



50









(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References































































(a) (b) (c)

(d) (e) (f)

(g) (h)


50 34 120583gmL) (c)



50 23 120583gmL) (g)













5 Conclusion




Acknowledgments


References
































































5 Conclusion




Acknowledgments


References






























































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