Antimutagenic Activities of Anisosciadium lanatum Extracts Could Predict the Anticancer Potential in Different Cell Lines

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1 Avilble online on Interntionl Journl of Phrmcognosy nd Phytochemicl Reserch 2017; 9(2); DOI number: /phyto.v9i Reserch Article ISSN: Antimutgenic Activities of Anisoscidium lntum Extrcts Could Predict the Anticncer Potentil in Different Cell Lines Wel M El-Syed 1,2*, Wrd A Hussin 3, Ahmd A Mhmoud 1,4, Mohmed A AlFredn 1 1 King Fisl University, Fculty of Science, Deprtments of Biologicl Sciences nd Chemistry, Al-Hufof 31982, Ahs, KSA. 2 University of Ain Shms, Fculty of Science, Deprtment of Zoology, Abbssi 11566, Ciro, Egypt 3 Al-Azhr University, Fculty of Science, Deprtment of Botny nd Microbiology, Ciro, Egypt. 4 Mini University, Fculty of Science, Deprtment of Chemistry, El-Mini, Egypt. Received: 24 th Dec, 16; Revised:18 th Jn, 17; Accepted:16 th Feb, 17; Avilble Online: 25 th Februry, 2017 ABSTRACT In spite of the tremendous progress in the development of nticncer drugs yet the cncer is still one of the leding cuses of deth worldwide in ddition to the economic nd socil burdens it cuses. The high cost of chemotherpy, the resistnce development nd the severe dverse effects mndte the continuous screening for novel, chep, nd sfe nticncer drugs. In our efforts to expedite the screening process in botnicl extrcts, we nlyzed the flvonoid, lycopene, β-crotene, nd chlorophyll nd b in four different extrcts from Anisoscidium lntum with different polrities in ddition to the essentil oil. In ddition, we hve lso mesured the ntioxidnt, peroxide nd superoxide scvenging ctivities of the extrcts nd oil. We hve lso estimted the ntimutgenic ctivities of these extrcts in Slmonell typhimurium using Ames test ginst two mutgens; sodium zide (NN 3) nd benzo()pyrene (B()P). All these fetures nd mesurements of the extrcts were correlted with their nticncer ctivities in 5 cell lines; liver, lung, colon, brest, nd prostte. The phytochemicl nd ntioxidnts studies did not precisely predict the potentil nticncer ctivity of extrcts or t lest wht is performed in the current study. Collectively, the ntimutgenic ctivities of the extrcts long with the reductions in mutnt frequency reported correltes well with the nticncer ctivities. Therefore, we believe tht the ntimutgenic ctivity long with phytochemicl nlysis could serve s plusible surrogte in the prediction of potentil nticncer ctivity in the process of screening botnicl extrcts. Keywords: Anisoscidium lntum, ntimutgenicity, phytochemistry, nticncer, scvenging ctivity, cell lines. INTRODUCTION The number of cncer cses rises nnully nd so do the socil nd economic burdens. The 13.3 million new cses of cncer dignosed worldwide in 2010 cost 290 billion dollrs. The 21.5 million new cncer cses nticipted in 2030 re projected to cost 458 billion dollrs. In 2012 lone, it is estimted tht 8.2 million died of the disese 1. Chemotherpy remins one of the stndrd therpies used to destroy cncer cells. However, it cn lso hrm helthy rpidly growing norml cells resulting in serious side effects 2. Chemotherpy my cuse secondry cncer or drive cncer into greter mlignncy. The cure rtes re still extremely bd for mny cncers nd mny cncer cell types develop resistnce to the chemotherpy regimens in use 3. All of the previous fcts mke the serch for novel drugs precedence gol for reserch. These novel ntitumour drugs should cuse fewer side-effects, cheper, nd hve greter therpeutic efficiency. Vrious plnt products hve been reported to possess chemopreventive properties. Approximtely, 20% of cncer incidents re preventble by consuming more vegetbles nd fruits which my potentilly prevent pproximtely 200,000 cncer-relted deths nnully 4. The drug discovery of novel ntitumor gents usully begins with screening for plnt extrcts with ntioxidnt nd ntimutgenic ctivities. This is usully followed by some in vitro nticncer studies nd finlly with time consuming niml studies. The ctive ingredients should be t some point isolted nd chemiclly chrcterized. Anisoscidium lntum Boiss is n edible plnt belonging to fmily Apicee. There re bout 3000 members of this fmily worldwide; minly in the temperte res. Some well-known vegetbles nd herbs belong to this fmily like crrot, prsnip, celery, fennel, ngelic, cumin, prsley, corinder/cilntro, Dill, nd crwy 5,6. The biologicl ctivity of this plnt hs not been studied before nd only the chemicl composition of the essentil oil ws reported in literture 7. Most members of this fmily re edible nd showed biologicl ctivity justifying the selection of this plnt for investigtion. Therefore, the im of the present study, consistent with our continuous effort to identify ntitumor plnts nd plnt frctions, ws to investigte the potentil ntioxidnt, ntimutgenic, nd nticrcinogenic ctivities of extrcts with different polrities from Anisoscidium lntum ginst rt heptic H4IIE1, humn colon HT-29, humn *Author for Correspondence: welelhlwny@hotmil.com

2 brest MCF-7, humn lung A549, or humn prostte PC3 cell lines. This study could offer pltform for future studies nd help selecting the key feture(s) of n extrct tht could most probbly identify the extrct with potentil cytotoxic nd nticncer ctivities. This will help lunch further niml studies nd isoltion nd chemicl chrcteriztion of the ctive gents from tht promising extrct. MATERIALS AND METHODS Chemicls nd preprtion of extrcts All regents were purchsed from Sigm (St. Louis, MO, USA) except where indicted. The eril prts of Anisoscidium lntum were collected from Al-Nyrri, Ajmn Vlley (Sudi Arbi), in April The plnt mteril ws identified by Prof. Mohmed Al Fredn nd voucher specimen (Anis-1-11) hs been deposited in the Deprtment of Biologicl Sciences, College of Science, King Fisl University, Sudi Arbi. The collected plnt mterils were stored in dry nd drk plce t room temperture with pssive ventiltion for 2 weeks. Extrction of the plnts ws performed using continuous technique. The eril prts (1.2 Kg) of Anisoscidium lntum Boiss, were dried nd milled. The powder of the plnt ws extrcted with methyelene chloride (CH 2Cl 2) nd methnol (MeOH) (1:1) t room temperture for 3 dys nd filtered. The solvents were evported under vcuum. Also, the essentil oil of Anisoscidium lntum ws isolted through Clevenger Apprtus. The extrct ws concentrted using vcuum rotry evportor nd stored t 4 C for chemicl nd biologicl studies. The dry extrcts were subjected to generl frctiontions using different orgnic solvents (n-hexne, CH 2Cl 2, nd MeOH, nd their mixtures) ccording to the chemicl nture of the components which they might contin (Tble I). The extrcts nd frctions were subjected to the usul isoltion nd purifiction processes; prtition between solvents, chromtogrphy (Thin Lyer Chromtogrphy (TLC), Grvity Column Chromtogrphy (CC) over Silic Gel, Diion, Sephdex LH-20 nd Polymide, nlyticl nd Flsh Chromtogrphy). The S9 mix used in ntimutgenicity study consisted of filter-sterilized NADPH (1.25 mm) nd heptic S9 frction (4 mg protein/ml) prepred from mle Sprgue Dwley rts treted with single dose (25 mg/kg, i.p. in corn oil) of Aroclor Cell cultures nd bcteri The cell lines used were heptic rt H4IIE cells, humn colon HT29, brest MCF7, lung A549, nd prostte PC3. All cells, medi, fetl bovine serum, DMSO, nd trypsin- EDTA were obtined from Americn Type Culture Collection (ATCC; Mnsss, VA, USA). Cell lines were seeded in 75-cm 2 tissue culture flsks t 37ºC in humidified tmosphere (5% CO 2) nd the medium ws renewed every two dys. Determintion of totl flvonoids Totl flvonoids content ws determined s described elsewhere 8. A stndrd curve for quercetin ws estblished nd the dt re expressed s mg quercetin equivlents/g dried plnt mterils. Assys were performed in triplicte. Determintion of β-crotene, lycopene, nd Chlorophyll nd b β-crotene, lycopene, nd chlorophyll nd b were determined s described by Ngt nd Ymshit 9. Contents of β-crotene, lycopene, nd chlorophyll nd b were determined ccording to the following equtions: lycopene (mg/100 ml) = A A A A 453 β-crotene (mg/100 ml) = A A A A 453 Chlorophyll (mg/100 ml) = A A 645 Chlorophyll b (mg/100 ml) = A A 645 The results re expressed s mens ± stndrd error of the men (SEM). The vlues re expressed s mg/g extrct. Assys were performed in triplicte. Determintion of totl ntioxidnt ctivity The ntioxidnt cpcity of Anisoscidium extrcts ws mesured spectrophotometriclly using phosphomolybdenum method bsed on the reduction of Mo (VI) to Mo (V) nd the subsequent formtion of specific green phosphte / Mo (V) compounds 10. Stock solutions of trolox were prepred in methnol just before use. The totl ntioxidnt ctivity ws expressed s equivlents of trolox (μg trolox/g extrct). Assys were performed in triplicte. Peroxide scvenging ctivity Peroxide scvenging ctivity ws mesured s described elsewhere 11. Peroxide rdicls were generted from mixing FeSO 4 nd H 2O 2. The peroxide scvenging ctivity (%)= [1 - (A 1 - A 2)/A o] x 100 where A 0 is the bsorbnce of the control (without extrct or trolox) nd A 1 is the bsorbnce including the extrct or trolox, nd A 2 is the bsorbnce without sodium slicylte. Assys were performed in triplicte. Superoxide nion scvenging ctivity Superoxide nion scvenging ctivity of the extrcts ws determined s described elsewhere 12. Superoxide rdicls were generted in phenzine methosulfte (PMS)- nicotinmide denine dinucleotide (NADH) systems nd ssyed by the reduction of nitroblue tetrzolium (NBT). The inhibition percentge of superoxide nion genertion ws clculted using the following formul: Inhibition of superoxide nion (%) = [(A 0 - A 1)/A 0 x 100] where A 0 is the bsorbnce of control, nd A 1 is the bsorbnce in presence of either extrct or trolox. Assys were performed in triplicte. Metl chelting ctivity Plnt extrct or stndrd ntioxidnt (0-600 µg/ml) ws dded to 50 µl of 2 mm FeCl 2. The rection ws initited by the ddition of 5 mm ferrozine (0.2 ml) nd the mixture ws shken vigorously t room temperture for 10 min 13. The bsorbnce ws mesured t 562 nm. The metl chelting ctivity ws clculted s follows: Metl chelting effect (%) = [(A 0 -A 1 )/A 0 x 100] where A 0 is the bsorbnce of control nd A 1 is the bsorbnce in presence of either extrct or stndrds (trolox or α-tocopherol). Assys were performed in triplicte. IJPPR, Volume 9, Issue 2: Februry 2017 Pge 198

3 Free rdicl scvenging ctivity The free rdicl scvenging ctivity of Anisoscidium extrcts ws evluted using 2,2-diphenyl-1-picryhydrzyl (DPPH ) 14. DPPH scvenging effect (%) = [(A 0 -A 1)/A 0 x 100] where A 0 is the bsorbnce of control nd A 1 is the bsorbnce in presence of either extrct or α-tocopherol. Cytotoxicity ssy on Slmonell typhimurium (TA1535) The cytotoxicity ssy ws performed using the bcteril growth ssy on nutrient gr pltes 15. The experiment ws designed with conditions tht mimic those of the revertnt mutgenesis/ntimutgenesis ssy. In preliminry experiment, 10-7 dilution of TA1535 ws shown to be the pproprite dilution; therefore, it ws selected to proceed with. Briefly, 100 µl of 10 7 dilution of the overnight growing bcteril culture in Luri Broth (LB) medium ws incubted with ech plnt extrct (5, 10, or 20 mg/ml) nd mixed with 2.5 ml of wrm 0.6% top gr (NCl/gr). The mix ws then dded to nutrient gr pltes nd the pltes were incubted t 37 C for 24 hours. After the incubtion period, colonies on triplicte pltes were counted nd compred to control pltes contining no plnt extrcts. Concentrtions investigted from herefter were 1 nd 5 mg for ll extrcts. Cytotoxicity of plnt extrcts in combintion with the selected mutgen To rule out ny possible toxic effect resulting from combintion of the mutgen used nd plnt extrcts in the mutgenicity/ntimutgenicity evlution ssys, the number of colony/plte evlution ws performed. A 100 μl of 10-7 dilution of overnight growing S. typhimurium TA1535 ws incubted with ech extrct t 37 C for 30 minutes on shking incubtor in the presence of 2 μg/plte of NN 3 or 20 μm of B[]P nd S9 mix in 400 μl of phosphte buffer. The mix ws then dded to gr pltes nd incubted t 37 C nd the totl vible bcteril count on triplicte pltes ws recorded fter 24 hours 16. Control experiments were crried out simultneously; the S9 mix lone hd no effect on the bcteril vibility. Mutgenicity nd Antimutgenicity Assys The reverse bcteril muttion ssy ws performed for screening the mutgenic potentil of the plnt extrcts s described elsewhere Spontneous revertnt colonies rise on pltes contining neither mutgens nor extrcts were counted. Revertnt colonies seen with 20 μm B[]P with S9 mix were performed s positive control. All ssys were performed in triplictes. Antimutgenic ctivity of Anisoscidium lntum extrcts ginst NN 3 or B[]P ws determined under pre-exposure nd coexposure ssys s described elsewhere In ll ssys, positive nd negtive controls were performed. All ntimutgenesis determintions were performed in triplictes. Revertnt colonies were counted fter hours of incubtion nd the ntimutgenic potentil of the tested extrcts ws expressed s percentge of reduction in mutgenicity 19, nd clculted ccording to the following eqution: % Reduction in mutgenicity = ([Rm-Rs] - [R - Rs] / [Rm Rs]) x100 Where Rm is the number of revertnts /plte in the presence of mutgen Rs is the number of spontneous revertnts /plte R is the number revertnts /plte in the presence of plnt extrct. A 20% or less reduction mens no ntimutgenic ctivity, % reduction mens moderte ctivity, nd 40 % or more reduction mens strong ntimutgenic ctivity. The mutnt frequency or muttion rte ws then clculted from the mutnt colonies/vible colonies for both exposure conditions for the mutgens investigted. Determintion of in vitro nticncer ctivity The nticncer ctivity ws determined s described elsewhere The cell lines were grown in the suitble medium. The cells were inoculted into 96 well pltes t plting densities of ~ 5,000 cells/well, four wells were used for ech tretment. Since the extrcts re colored, control wells (extrct control) were mde for every extrct concentrtion used without cells. Three independent experiments were performed. Percentge growth inhibition ws clculted using the following formul: [(T-T0)/(C-T0)] x 100 for concentrtions for which T T0 [(T-T0)/T0] x 100 for concentrtions for which T < T0. T0 or time zero represents mesurement of the cell popultion for ech cell line t the time of extrct ddition, C is the control growth, nd T is the test growth t different concentrtions of Anisoscidium extrcts fter incubtion. Three dose response prmeters were clculted for ech experimentl gent. Growth inhibition of 50 % (GI50) ws clculted from [(T-T0)/(C-T0)] x 100 = 50, which is the extrct concentrtion resulting in 50% reduction in the net protein increse (s mesured by SRB stining) in control cells during the incubtion. The extrct concentrtion resulting in totl growth inhibition (TGI) is clculted from Ti = Tz. The LC50 (concentrtion of extrct resulting in 50% reduction in the mesured protein t the end of the extrct tretment s compred to tht t the beginning) indicting net loss of cells following tretment is clculted from [(Ti-Tz)/Tz] x 100 = -50. All procedures were pproved by the University of King Fisl Committee of Scientific Reserch Ethics. Sttisticl nlysis of dt Sttisticl nlyses were performed using ANOVA, followed by Fisher s protected lest significnt difference multiple rnge test. Differences were considered significnt t P < RESULTS AND DISCUSSION Phytochemistry, ntioxidnt nd rdicl scvenging ctivities Extrction of Anisocidium ws performed using solvents with different polrities. Extrct I (n-hexne extrct) would presumbly contin the highly non-polr constituents, extrcts II (methylene chloride: methnol: petroleum ether) nd III (wter: methnol) would contin intermedite polr flvonoids nd glycosides nd some other polr compounds. Extrct IV (wter extrct) would hve the highly polr constituents such s glycosides, polr flvonoids nd glycones. Extrct V is the essentil oil. IJPPR, Volume 9, Issue 2: Februry 2017 Pge 199

4 Tble 1: Extrction Scheme of Anisoscidium lntum. Smple Code Extrction Solvent B1 (I) n-hexne 100% B2 (II) CH 2Cl 2:MeOH:Petrolem ether (1:1:1) B3 (III) H 2O:MeOH (1:1) B4 (IV) H 2O 100% B5 (V) Essentil oil Extrct II hs moderte flvonoid content equivlent to ~ 25 mg quercetin / g. Extrcts III nd IV nd essentil oil hve low flvonoid content (Tble 1), while extrct I is lmost devoid of flvonoids. With the exception of extrct I which is very poor in lycopene, ll other extrcts nd essentil oil hve moderte lycopene content. Extrct II hs the highest lycopene content of ~ 41 mg/g followed by extrct III which hs 30 mg/g lycopene content (Tble 1). The β-crotene content ws higher in essentil oil (31 mg/g) nd polr extrcts III (31 mg/g) nd IV (27 mg/g) thn in more non-polr extrcts I nd II. The chlorophyll nd chlorophyll b content vried with the extrction solvent. Extrcts I, II, nd III hve more chlorophyll content thn extrct IV nd essentil oil (Tble 1). Extrcts II nd III hve the highest contents of chlorophyll nd b. Extrct I hs the highest metl chelting ctivity comprble to tht of tocopherol, followed by extrcts III >> II >> IV. The ctivity of ll extrcts ws concentrtiondependent (Figure 1). The essentil oil (B5) hd the lowest metl chelting ctivity. Extrcts II nd III hd the highest free rdicl scvenging ctivity followed by extrct IV nd essentil oil. Extrct I hd the lowest ctivity (Figure 2). Only extrct III (wter: methnol) with the high β- crotene, chlorophyll nd b content resulted in significnt totl ntioxidnt ctivity equivlent to ~ 101 μg trolox/g extrct. It lso hd significnt peroxide nd superoxide scvenging ctivities (Tble 2). Extrct IV rich only in β-crotene resulted in significnt superoxide scvenging ctivity s compred to trolox. All plnt extrcts showed peroxide scvenging ctivity higher thn tht of trolox nd the highest ctivity of ~ 99% ws shown by the essentil oil compred to 32% shown by trolox (Tble 2). Extrcts III nd IV hve the highest superoxide scvenging ctivity (Tble 2). From our previous study on Conyz trilob, we found tht high chlorophyll content ws determining fctor nd hd strong correltion with the cytotoxic ctivity of extrcts ginst tumor cells 16. With n exception of the effect of extrct I in the present study, extrcts II nd III rich in chlorophyll, flvonoids, lycopene, nd β-crotene showed the best ntioxidnt nd scvenging ctivities ginst peroxide nd superoxide rdicls. These extrcts lso showed the best nticncer ctivities. No doubt the oxidtive stress is key event during the pthogenesis of mny diseses nd during the initition nd progression of cncer 21. However, extrct I (hexn extrct) poor in ll the previous constituents ws s potent s extrcts II nd III in ntimutgenic nd nticncer studies. Therefore, the phytochemistry performed in the current study lone is not vlid tool to build pltform for identifiction of plnt extrcts with potentil nticncer ctivities. The only ntioxidnt/scvenging property tht chrcterized this highly non-polr extrct I ws its unique metl chelting ctivity. Li et l found tht the hexne extrct of Gynostemm pentphyllum rrested the cell cycle, inhibited the cncer cells nd induced poptosis in brest cell lines 22. Although most studies focus on polr extrcts, but non-polr extrcts re lso promising nd should not be neglected. Alkloids, esters nd terpenes could be found in hexne extrct nd mny of these were reported to hve nticncer ctivity Antimutgenic ctivity Mesuring the vibility of bcteri nd estimting nontoxic concentrtion is pre-requisite for the vlidity of Ames ssy results. The vibility of the Slmonell typhimurium ws not ffected by either 5 or 10 mg/ml (Tble 3). All extrcts t 20 mg/ml significntly reduced the bcteril vibility. Extrcts II nd V were the most toxic extrcts reducing the vibility by 93 nd 94%, respectively (Tble 3). The effect of combintion of NN 3 or B[]P/S9 mix with plnt extrcts t 1 or 5 mg ws investigted nd ws shown to hve no effect on the bcteril vibility (Tbles 4 nd 5). Extrcts I-IV hd no mutgenic ctivity when compred with B[]P. Essentil oil ws mutgenic to the bcteri t 5 mg/ml resulting in colonies number comprble to those produced fter B[]P (Tble 6). This essentil oil ws excluded from further studies. Similr properties were reported before for essentil oils from mint nd rosemry which should be crefully evluted nd used with gret cution 26 lthough we usully use concentrtions in the experimenttion higher thn those used by humns. Extrcts I, II, nd III exerted strong ntimutgenic ctivity ginst NN 3 in dose dependent mnner (Tble 7) nd regrdless of the type of exposure conditions. Extrct IV hd strong ntimutgenic ctivity ( 40%) but only under pre-exposure conditions t the high dose, where the extrct ws incubted with the bcteri before the ddition of NN 3 (Tble 7). In dose-independent mnner, ll extrcts hd strong ntimutgenic ctivity ginst Tble 1b: Totl flvonoids, lycopene, β-crotene, nd chlorophyll nd b content of different Anisoscidium lntum extrcts. Extrct Totl flvonoids (mg quercetin) Lycopene (mg/g) β-crotene Chlorophyll Chlorophyll b (mg/g) (mg/g) (mg/g) I 0.02 ± ± ± ± ± 0.4 II ± ± ± ± ± 4.2 III 8.70 ± ± ± ± ± 6.5 IV 2.87 ± ± ± ± ± 3.4 V 0.20 ± ± ± ± ± 1.9 The dt re expressed mens ± SEM. All ssys were performed in triplictes. IJPPR, Volume 9, Issue 2: Februry 2017 Pge 200

5 Tble 2: Totl ntioxidnt, peroxide scvenging, nd superoxide scvenging ctivities of vrious Anisoscidium lntum extrcts. Antioxidnt ctivity,b Peroxide scvenging ctivity Superoxide scvenging ctivity (%) (%) I 54.3 ± ± ± 2.0 II 28.7 ± ± ± 6.3 III ± ± ± 9.4 IV 64.3 ± ± ± 6.5 V 65.0 ± ± ± 2.7 Trolox ± ± ± 3.7 The dt re expressed s mens ± SEM, n = 3. b The ctivity is expressed s equivlent of trolox (µg trolox /g extrct). Tble 3: Effect of vrious Anisoscidium lntum extrcts (I-V) on Slmonell typhimurium TA1535 vibility s ssessed by colony formtion on plte. Vibility t extrct concentrtions (colonies/plte (% of control)) 5 mg/ml 10 mg/ml 20 mg/ml None 31.7 ± 4.2 (100) I 35.3 ± 4.5 (111) 27.0 ± 2.5 (85) 4.7 ± 1.0 (15) II 39.0 ± 2.9 (123) 28.0 ± 1.8 (88) 2.3 ± 0.7 (7) III 41.7 ± 5.1 (132) 30.3 ± 3.1 (96) 13.0 ± 2.1 (41) IV 33.3 ± 2.7 (105) 31.7 ± 2.5 (100) 16.3 ± 1.3 (51) V 29.7 ± 3.0 (94) 26.0 ± 2.2 (82) 2.0 ± 0.1 (6) Significntly different (p < 0.05) from the number of colonies/plte (men ± SEM) recorded in the bsence of plnt extrcts. Assys were performed in triplictes. Tble 4: Effect of Anisoscidium lntum extrct nd sodium zide (NN 3) on Slmonell typhimurium TA1535 vibility s ssessed by colony formtion on plte. Vibility t NN 3 nd extrct concentrtions (colonies/plte (% of control)) 1 mg/ml 5 mg/ml None 41.0 ± 2.9 (100) NN 3 (2 µg/plte) 43.3 ± 3.3 (106) I 39.3 ± 4.0 (96) 39.0 ± 4.3 (95) II 38.7 ± 2.5 (94) 37.3 ± 2.9 (91) III 40.0 ± 3.7 (98) 39.7 ± 4.1 (97) IV 44.0 ± 3.9 (107) 38.7 ± 4.5 (94) V 39.7 ± 3.0 (97) 40.0 ± 3.0 (98) Dt re expressed s men ± SEM. Assys were performed in triplictes. B[]P under both exposure regimens investigted (Tble 8). Extrcts I-III significntly reduced the NN 3-mutnt frequency by 64-94%. Extrct IV resulted in significnt reduction in NN 3-mutnt frequency (22-70%) only under pre-exposure conditions (Tble 9). All extrcts significntly reduced the B[]P-mutnt frequency by 52-83% (Tble 10). Muttion is n essentil event in the pthogenesis of cncer nd tumor formtion. Ames test is used worldwide with gret success to detect mutgenic chemicls It is very simple ssy with low cost nd high efficiency nd ccurcy compred with other ssys. S. typhimurium TA1535 strin contins the bse-pir substitution muttion Tble 5: Effect of Anisoscidium lntum extrcts, benzo[]pyrene (B[]P), nd S9 mix on Slmonell typhimurium TA1535 vibility s ssessed by colony formtion on plte. Vibility t B[]P nd plnt extrct concentrtions (colonies/plte (% of control)) 1 mg/ml 5 mg/ml None 30.0 ± 2.7 (100) B[]P (20 µm) 34.0 ± 3.3 (113) I 31.3 ± 3.0 (104) 26.7 ± 2.6 (89) II 25.0 ± 2.2 (83) 23.7 ± 0.7 (79) III 29.7 ± 2.7 (99) 24.0 ± 1.7 (80) IV 36.3 ± 3.5 (121) 25.7 ± 2.7 (86) V 28.7 ± 1.8 (96) 24.3 ± 2.0 (81) Dt re expressed s men ± SEM. Assys were performed in triplictes. hisg46 which is known to be more responsive to sodium zide thn other direct mutgens 16. We hve chosen two different mutgens. NN 3 which is direct intercltion gent tht needs no prior metbolism nd B()P which requires metbolic mchinery offered by NADPH/S9 mix. This could help in the understnding of the ntimutgenic ctivities of the plnt extrcts. We lso performed the ntimutgenicity study with two types of exposure; in pre-exposure the bcteri were incubted with the extrct before the ddition of the mutgen nd coexposure where the bcteri, extrcts nd mutgens were ll dded t the sme time. Since the pre-exposure results for B()P (verge 96%) were bit better thn the co- IJPPR, Volume 9, Issue 2: Februry 2017 Pge 201

6 Tble 6: Determintion of mutgenic ctivity of Anisoscidium lntum extrcts in Slmonell typhimurium TA1535 in presence of S9 mix. Plnt extrcts mutgenicity (revertnt colonies/plte; men ± SEM) 1 mg/ml 5 mg/ml None (spontneous) 25.7 ± 4.0 B[]P (20 µm) ± 14.6 I 10.0 ± ± 1.8 II 14.0 ± ± 2.3 III 18.0 ± ± 1.9 IV 11.0 ± ± 1.2 V 74.7 ± ± 11.5 Significntly different (p < 0.05) from non-treted bcteri (spontneous muttions). Assys were performed in triplictes. exposure (verge of 82%). We my ssume tht extrcts could interfere with the metbolic bioctivtion of B()P by inhibiting to some extent CYP4501A1 responsible for the ctivtion of the mutgen. However, this could be prtly true but the 82% reported in co-exposure nd the indiscrimintion seen in the ntimutgenic ctivity of extrcts ginst NN 3 suggests nother mechnism. One such plusible mechnism could be the direct binding nd protection of DNA from the electrophilic mutgens or metbolites 27. Another mechnism could be the elevtion in the ntioxidnts milieu of the cells thus, promoting the DNA repir systems 28. Anticncer ctivity Extrcts II nd III showed the most effective nticncer ctivity with GI50 t concentrtion of s low s ng ginst liver, lung, brest, nd prostte cell lines used (Tble 11). Extrct I ws less effective with GI50 of ng. The colon cells were the lest ffected nd only extrct IV ws ble to inhibit the growth of cells t very low concentrtion (60 ng). Extrcts I-III chieved totl growth inhibition (TGI) of liver, lung, nd brest cells t ng (Tble 11b). Extrcts II nd III showed very similr pttern ginst prostte cells nd hd TGI vlues t 560 nd 480 ng, respectively. Extrct IV ws very specific in ffecting only the resilient colon cells, which hd not been significntly ffected by other extrcts nd hd TGI vlue of 380 ng. All extrcts needed high concentrtions to kill 50% of the cells initilly present t the time of the ddition of the extrct (LC50). LC50 vlues recorded for brest, liver, prostte, colon, nd lung cells were 100 μg. Extrct III hd low LC50 vlues of ~ 51 nd 48 in liver nd lung cells, respectively (Tble 11c). The NCI screened more thn 50,000 plnt extrcts nd sets up cutoff vlue for potentil nticncer extrct t 10µg Since the current dt showed tht the plnt extrcts under study hve their GI50 t the nnogrm rnge, so these extrcts re considered highly ctive nd Tble 7: Determintion of nti-mutgenic ctivity of Anisoscidium lntum extrcts in Slmonell typhimurium TA1535 ginst sodium zide (NN 3; 2 µg/plte). NN 3 mutgenicity t plnt extrct concentrtions (revertnt colonies/plte; men ± SEM (% reduction in NN 3 mutgenicity)) Pre-exposure * Co-exposure * 1 mg/ml 5 mg/ml 1 mg/ml 5 mg/ml NN ± 17.1 (0) I 77.7 ± 6.3 (78) 14.7 ± 1.2 (101),b 21.7 ± 3.6 (98) 10.7 ± 2.2 (102) II 50.0 ± 5.8 (88) 13.3 ± 1.5 (101),b 54.0 ± 7.1 (86) 21.7 ± 5.6 (98),b III ± 10.8 (4) 27.7 ± 9.3 (96),b 97.7 ± 11.4 (70) 17.0 ± 2.9 (100),b IV ± 25.8 (22) 77.0 ± 9.3 (78),b ± 20.9 (1) ± 12.0 (6) * Plnt extrcts were incubted with bcteri either 30-minutes before NN 3 (Pre-exposure) or incubted with the bcteri nd NN 3 (co-exposure). The spontneous revertnt colonies were 17.0 ± 2.1. Significnt (p < 0.05) reduction (% of inhibition of mutgenicity indicted in prentheses) from revertnt colonies seen with NN 3. b Significnt difference (p < 0.05) between plnt extrct concentrtions. Tble 8: Determintion of nti-mutgenic ctivity of Anisoscidium lntum extrcts in Slmonell typhimurium TA1535 ginst benzo[]pyrene (B[]P; 20 µm) in presence of S9 mix. B[]P mutgenicity t plnt extrct concentrtions (revertnt colonies/plte; men ± SEM (% reduction in B[]P mutgenicity)) Pre-exposure * Co-exposure * 1 mg/ml 5 mg/ml 1 mg/ml 5 mg/ml B[]P ± 21.5 I 52.0 ± 2.5 (97) 53.0 ± 5.5 (97) ± 9.3 (74) ± 6.8 (73) II 64.0 ± 4.4 (93) 55.0 ± 5.7 (96) 86.3 ± 3.5 (85) 81.0 ± 8.5 (87) III 66.0 ± 3.8 (92) 51.0 ± 5.5 (98) 86.0 ± 5.1 (85) 85.7 ± 5.6 (85) IV 63.3 ± 5.4 (93) 49.7 ± 4.4 (98) 81.7 ± 10.3 (87) 85.0 ± 5.1 (86) * Plnt extrcts were incubted with bcteri either 30-minutes before B[]P (Pre-exposure) or incubted with the bcteri nd B[]P (co-exposure). The spontneous revertnt colonies were 45.0 ± 4.9. Significnt (p < 0.05) reduction (% of inhibition of mutgenicity indicted in prentheses) from revertnt colonies seen with B[]P. IJPPR, Volume 9, Issue 2: Februry 2017 Pge 202

7 Tble 9: Effects of Anisoscidium lntum extrcts on sodium zide (NN 3) mutnt frequency. Mutnt frequency nd (% of NN 3) # Pre-exposure * Co-exposure * 1 mg/ml 5 mg/ml 1 mg/ml 5 mg/ml NN (100) I 1.98 (30) 0.38 (6) 0.55 (8) 0.27 (4) II 1.29 (19) 0.36 (5) 1.40 (21) 0.58 (9) III 7.02 (105) 0.70 (10) 2.44 (36) 0.43 (6) IV 5.23 (78) 1.99 (30) 6.56 (98) 7.06 (105) # Clculted from mutnt colonies (tble 8)/ vible colonies (tble 5). * Plnt extrcts were incubted with bcteri either 30-minutes before NN 3 (Pre-exposure) or incubted with the bcteri nd NN 3 (co-exposure). Significnt difference (p < 0.05) from NN 3. Tble 10: Effects of Anisoscidium lntum extrcts on benzo[]pyrene (B[]P) mutnt frequency. Mutnt frequency nd (% of B[]P) # Pre-exposure * Co-exposure * 1 mg/ml 5 mg/ml 1 mg/ml 5 mg/ml B[]P 9.54 (100) I 1.66 (17) 1.99 (21) 3.76 (39) 4.54 (48) II 2.56 (27) 2.32 (24) 3.45 (36) 3.42 (36) III 2.22 (23) 2.13 (22) 2.90 (30) 3.57 (37) IV 1.74 (18) 1.93 (20) 2.25 (24) 3.31 (35) # Clculted from mutnt colonies (tble 9)/ vible colonies (tble 6). * Plnt extrcts were incubted with bcteri either 30-minutes before B[]P (Pre-exposure) or incubted with the bcteri nd B[]P (co-exposure). Significnt difference (p < 0.05) from B[]P. Tble 11: The 50% growth inhibition (GI50) dt of different Anisoscidium lntum extrcts in cell lines. Extrct Potency of extrcts (µg) in cell lines (men ± SEM), n = 5 H4IIE1 A549 HT29 MCF7 PC3 I 0.16 ± ± ± ± ± 0.11 II 0.09 ± ± 0.01 >> ± ± 0.01 III 0.08 ± ± ± ± ± 0.01 IV 1.28 ± ± ± ± ± 0.04 Tble 11b: The totl growth inhibition (TGI) dt of different Anisoscidium lntum extrcts in cell lines. Extrct Potency of extrcts (µg) in cell lines (men ± SEM), n = 5 H4IIE1 A549 HT29 MCF7 PC3 I 0.70 ± ± ± ± ± 1.02 II 0.58 ± ± 0.03 >> ± ± 0.06 III 0.42 ± ± ± ± ± 0.06 IV 8.56 ± ± ± 0.02 > ± 0.15 Tble 11c: The 50% lethlity (LC50) dt of different Anisoscidium lntum extrcts in cell lines. Extrct Potency of extrcts (µg) in cell lines (men ± SEM), n = 5 H4IIE1 A549 HT29 MCF7 PC3 I ± > 100 > 100 > 100 > 100 II ± 9.54 > 100 >> 100 > ± III ± ± 6.04 > 100 > ± 7.07 IV > 100 > ± 8.94 >> 100 > 100 extrpolted from dose-response curve. H4IIE1 (rt liver), A549 (humn lung), HT29 (humn colon), MCF7 (humn brest), PC3 (humn prostte). GI50 is the concentrtion of n extrct (µg) tht cuses 50% growth inhibition. TGI is the concentrtion of n extrct (µg) tht yields no net growth over the course of the ssy. LC50 is the concentrtion of n extrct (µg) tht kills 50% of the cells tht were present t the time of the ddition of the extrct. worthy of further investigtions. One other unique feture tht mkes these extrcts promising is their sfety which is evident from LC50 dt. Hving LC50 t much higher concentrtions wy from those needed for GI50 mens these extrcts re sfe. It lso mens tht these extrcts inhibited the growth of the cncer cells rther thn merely IJPPR, Volume 9, Issue 2: Februry 2017 Pge 203

8 Inhibition % Inhibition % Wel et l. / Antimutgenic Activities of B1 B2 B3 B4 B5 Tocopherol Trolox Conc. (µg/ml) Figure 1: Metl chelting ctivity of Anisoscidium lntum extrcts. B1 B2 B3 B4 B5 Tocopherol Conc. (µg/ml) Figure 2: Free rdicl scvenging ctivity of Anisoscidium lntum extrcts. killing the cells. Modultion of cncer cell signling, nd triggering poptosis nd cell cycle rrest could be held responsible for the reported nticncer ctivities of the extrcts but these ssumptions need further studies for confirmtion. Extrct IV with highly polr compounds discerned itself by cting specificlly ginst colon cells which cnnot be explined but other studies showed tht highly polr glycosides, glycons nd flvonoids trget colon HT29 with specificity nd efficcy 29. CONCLUSIONS The n-hexne extrct showed remrkble metl chelting nd nticncer ctivities. This extrct will be frctionted nd the ctive constituents will be identified. The nonpolr constituents deserve more ttention. The phytochemicl nd ntioxidnts studies did not precisely predict the potentil nticncer ctivity of extrcts or t lest wht is performed in the current study. Collectively, the ntimutgenic ctivities of the extrcts long with the reductions in mutnt frequency reported correltes well with the nticncer ctivities. Therefore, we believe tht the ntimutgenic ctivity long with phytochemicl nlysis could serve s plusible surrogte in the IJPPR, Volume 9, Issue 2: Februry 2017 Pge 204

9 prediction of potentil nticncer ctivity in the process of screening botnicl extrcts. ACKNOWLEDGEMENT This project ws supported by King Abdulziz City for Science nd Technology (KACST), Grnt AT CONFLICT OF INTERESTS None declred. AUTHOR CONTRIBUTION AA nd MA identified nd collected the plnts nd performed the extrction. WE nd WH plnned nd crried out ll biologicl experiments. WE performed the sttisticl nlyses nd drfted the mnuscript. REFERENCES spx?Pge= eduction/wyntk-liver-cncer. 3. Demin AL, Vishnv P Nturl products for cncer chemotherpy. Microbil Biotechnol, 4: Prtheeshkumr P, Sreekl C, Zhng Z, Budhrj A, Ding S, Son K, Wng X, Hitron A, Hyun-Jung K, Wng L, Lee J, Shi X Cncer prevention with promising nturl products: mechnisms of ction nd moleculr trgets. Anti-Cncer Agents Med Chem, 12: Mulidini A, Fridh K, Alfi S, Khozirh L, Nordin H Chemicl chrcteriztion nd ntioxidnt ctivity of three medicinl Apicee species. Industril Crops nd Products, 55: Mggi F, Brboni L, Pp F, Cprioli G, Ricciutelli M, Sgrtini G, Vittori S A forgotten vegetble (Smyrnium olustrum L., Apicee) s rich source of isofurnodiene. Food Chem, 135: Al-Mzro SA Chemicl constituents of A. lntum, H. tubercultum, A. grcini, S. spinos, H. bcciferum, nd A. ludwigii grown in Sudi Arbi. J Sudi Chem Soc, 7: Chng CC, Yng MH, Wen HM, Chern JC Estimtion of totl flvonoid content in propolis by two complementry colorimetric methods. J Food Drug Anl, 10: Ngt M, Ymshit I Simple method for simultneous determintion of chlorophyll nd crotenoids in tomto fruit. Nippon Shokuhin Kogyo Gkkish, 39: Prieto P, Pined M, Aguilr M Spectrophotometric quntittion of ntioxidnt cpcity through the formtion of phosphomolybdenum complex: Specific ppliction to the determintion of vitmin E. Anl Biochem, 269: Smirnoff N, Cumbes QJ Hydroxyl rdicl scvenging ctivity of comptible solutes. Phytochem, 28: Nishikimi M, Appji N, Ygi K The occurrence of superoxide nion in the rection of reduced phenzine methosulfte nd moleculr oxygen. Biochem Biophys Res Commun, 46: Dinis TC, Mdeir VM, Almeid LM Action of phenolic derivtives (cetminophen, slicylte nd 5- minoslicylte) s inhibitors of membrne lipid peroxidtion s peroxyl rdicl scvenging effects. Chem Phrm Bull, 36: Blois MS Antioxidnt determintions by the use of stble free rdicl. Nture, 26: El-Syed WM, Hussin WA Antimutgenic nd Antioxidnt Activity of Novel 4-Substituted Phenyl- 2,2 -Bichlcophenes nd Az-Anlogues. Drug Des Devel Ther, 7: El-Syed WM, Hussin WM, Mhmoud AA, AlFredn MA The Conyz trilob Extrcts with High Chlorophyll Content nd Free Rdicl Scvenging Activity Hd Anticncer Activity in Cell Lines. Biomed Res Int, 2013: El-Syed WM, Hussin WA, Frnklin MR The nti-mutgenicity of 2-substitiuted selenzolidine-4- (R)-crboxylic cids. Muttion Res, 627: Mron DM, Ames BN Revised methods for the Slmonell mutgenicity test. Mutt Res, 113: Hong CE, Cho MC, Jng HA, Lyu SY Mutgenicity nd nti-mutgenicity of Acnthopnx divrictus vr lbeofructus. J Toxicol Sci, 36: Mns DR, d Roch AB, Schwrtsmnn G Anticncer drug discovery nd development in Brzil: trgeted plnt collection s rtionl strtegy to cquire cndidte nti-cncer compounds. Oncologist, 5: Li Y, Hung J, Lin W, Yun Z, Feng S, Xie Y, M W In Vitro Anticncer Activity of Nonpolr Frction from Gynostemm pentphyllum (Thunb.) Mkino. Evid-Bsed Compl Altern Med, 2016: Koduru S, Grierson DS, vn de Venter M, Afolyn AJ Anticncer Activity of Steroid Alkloids Isolted from Solnum culestrum. Phrmceut Biol, 45: Hung M, Lu JJ, Hung MQ, Bo JL, Chen XP, Wng YT Terpenoids: nturl products for cncer therpy. Expert Opin Investig Drugs. 21: Mchdo NF, Clheiros R, Fiuz SM, Borges F, Gspr A, Grrido J, Mrques MP Phenolic esters with potentil nticncer ctivity - the structurl vrible. J Moleculr Modeling, 13: Mistro EL, Mot SF, Lim EB, Bernrdes BM, Goulrt FC Genotoxicity nd mutgenicity of Rosmrinus officinlis (Lbite) essentil oil in mmmlin cells in vivo. Genet Mol Res, 9: Mrnewick JL, Gelderblom WC, Joubert E An investigtion on the ntimutgenic properties of South Africn herbl tes. Mutt Res, 471: Collins AR, Azquet A, Lngie SA Effects of micronutrients on DNA repir. Eur J Nutr, 51: IJPPR, Volume 9, Issue 2: Februry 2017 Pge 205

10 29. Antunes-Ricrdo M, Moreno-Grcí BE, Gutiérrez- Uribe JA, Aráiz-Hernández D, Alvrez MM, Sern- Sldivr SO Induction of poptosis in colon cncer cells treted with isorhmnetin glycosides from Opunti ficus-indic pds. Plnt Foods Hum Nutr, 69: IJPPR, Volume 9, Issue 2: Februry 2017 Pge 206