Extraction optimization of medicinally important metabolites from Datura innoxia Mill.: an in vitro biological and phytochemical investigation

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1 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 DOI /s RESEARCH ARTICLE Open Aess Extrtion optimiztion of mediinlly importnt metolites from Dtur innoxi Mill.: n in vitro iologil nd phytohemil investigtion Humir Ftim 1, Koml Khn 1, Muhmmd Zi 2, Tofeeq Ur-Rehmn 1, Bushr Mirz 3 nd Ihsn-ul Hq 1* Astrt Bkground: The present study ims to proe the impt of polrity dependent extrtion effiieny vrition on phrmologil spetrum of Dtur innoxi Mill. in order to reonnoiter its underexplored therpeuti potentil. Methods: A rnge of solvent extrts ws sujeted to phytohemil nd iologil ssys to find the most profiient solvent system nd plnt prt for eh type of iotivity. Totl phenoli nd flvonoid ontents were determined olorimetrilly nd speifi polyphenols were quntified y HPLC-DAD nlysis. The smples were iologilly evluted y employing multimode ntioxidnt, ytotoxi, protein kinse inhiition nd ntimiroil ssys. Results: Among ll the solvents used, mximum perent extrt reovery (33.28 %) ws otined in queous lef extrt. The highest mount of glli id equivlent phenoli nd queretin equivlent flvonoid ontent ws otined in the distilled wter nd ethyl ette-ethnol extrts of lef i.e., ± 0.12 nd ± 0.18 mg/g dry weight (DW) respetively. Reverse phse HPLC-DAD sed quntifition reveled the presene of signifint mounts of tehin, ffie id, pigenin nd rutin rnging from 0.16 to 5.41 mg/g DW. Highest DPPH rdil svenging tivity (IC 50 = μg/ml) ws displyed y the ethyl ette-etone stem extrt. Mximum totl ntioxidnt pity nd reduing power potentil were reorded in the queous lef nd ethyl ette stem extrts i.e., ± 0.24 nd ± 0.61 mg sori id equivlent/g DW respetively. Cytotoxiity ginst rine shrimps tegorized 25 % of the lef, 16 % of the stem nd 8.3 % of the fruit extrts s highly potent (LC μg/ml). Signifint ytotoxiity ginst humn leukemi (THP-1) ell line ws exhiited y the hloroform nd n-hexne fruit extrts with IC nd 3.49 μg/ml respetively. Ethyl ette nd methnol-hloroform extrts of lef nd stem exhiited onspiuous protein kinse inhiitory tivity ginst Streptomyes 85E strin with 22 mm ld phenotype. A noteworthy ntimiroil tivity ws exhiited y lef extrts ginst Miroous luteus nd n-hexne fruit extrt ginst Aspergillus niger (MIC 3.70 nd 12.5 μg/ml respetively). Conlusion: Multiple solvent system is ruil vrile to retrieve phrmologil potentil of mediinl plnts nd D. innoxi n e envisged s novel soure of nturl ntioxidnts, ntimiroils nd ntiner ompounds. Keywords: Dtur innoxi, Cytotoxiity, Protein kinse inhiition, THP-1 leukemi ell line * Correspondene: ihq@qu.edu.pk 1 Deprtment of Phrmy, Fulty of Biologil Sienes, Quid-i-Azm University, Islmd 45320, Pkistn Full list of uthor informtion is ville t the end of the rtile 2015 Ftim et l. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 2 of 18 Bkground Nturl produts from mediinl plnts, either s pure ompounds or stndrdized extrts provide indefinite prospets for new drug leds euse of the unmthed vilility of hemil diversity. Moderniztion of ethnomediinl plnt remedies through stndrdiztion nd qulity ontrol is key ftor tht will govern their widespred eptne y the interntionl ommunity. The hemo-diversity nd ure ll potentil of these herl ok-tils entute the role of nlysis using ethnophrmologil pproh s fundmentl [1]. Although the ulk of urrent knowledge of ethnomediinl plnts hs led to ountless improvements in helth re ut unfortuntely with the prompt industriliztion nd loss of ethni ultures some of this informtion will no dout dispper. The vlidtion of ethno otnil dt from the under explored folk plnt remedies undoutedly represent n inexhustile reservoir of novel moleules nd my led to innovtive strtegies for new drug disovery. One suh ethno mediinlly importnt plnt is Dtur innoxi Mill. (Solnee), the ntive rnge of whih ppers to e Chin, Mexio, United Sttes, Crien Islnds nd Asi. It is knowledged with ommon nme of Thorn-Apple, Downy Thorn-Apple nd Indin- Apple. It is low growing, spreding perennil with hiry 2 5 in. leves, white flowers nd spiny fruit. From nient times ontinuing to the present, its seeds re used in shmnisti rituls s pth to enlightenment [2]. The plnt onquers very speil ple in Ayurved sine ll plnt prts nmely flowers, leves, root, stem, fruit nd seeds hve een meritoriously employed for rnge of tretments suh s insnity, ries, leprosy, et. However, the indisriminte use of its extrt my use ute poisoning, delirium nd n even led to deth. The tive priniples in D. innoxi inlude tropine, sopolmine hyosymine, withnolides (ltones) nd other tropnes. A omprtive nlysis of the different seed extrts of vrious Dtur speies for their free rdil svenging pity towrds the stle DPPH rdil reveled tht D. innoxi hs the strongest ntioxidnt potentil [3]. Reently new withnolide, dinoxin B, isolted from the methnol extrt of D. innoxi leves demonstrted su-miromolr IC 50 vlues ginst multiple humn ner ell lines [4]. An exhustive review of literture led us to the onlusion tht the sientifi dt out the omplete phrmologil spetrum of D. innoxi is limited. There is no orgnized study yet onduted involving ttery of iossys on individul plnt prts tht ould demonstrte vried phrmologil potentil of this ethnomediinlly importnt plnt. The present study hs employed wide rnge of extrtion solvent polrity s vrile to demonstrte nd orrelte its effets on extrtion effiieny nd iotivity. To the est of our knowledge this is the first report highlighting plnt s potentil for the inhiition of vrious protein kinses nd its ytotoxi tivity ginst humn monoyti leukemi THP-1 ell lines. Sine iologil tivity is the leding thred of ethnophrmologil pproh nd hemo-profiling the distintive fingerprints for individul plnt prts ould e very enefiil for the development of uniform stndrdiztion tools. The present work is premeditted to evlute the ntioxidnt, ntifungl, ntiteril nd ytotoxi potentil of lef, stem nd fruit of D. innoxi in sientifi mnner y utilizing multipolrity extrtion system. Methods Plnt mteril olletion nd identifition The plnt mteril ws olleted in Septemer 2013 from Quid-i-Azm University Islmd. Identifition of the field olleted plnt ws uthentited s D. innoxi y Prof. Dr. Rizwn Aleem Qureshi, Deprtment of Plnt Sienes, Fulty of Biologil Sienes, Quid-i-Azm University Islmd, Pkistn. Dried vouher speimen ws rhived in the Herrium of mediinl plnts, Quid-i-Azm University Islmd under herrium numer PHM-487. Solvents nd regents All regents nd solvents used in the present study were of nlytil grde. Solvents for the extrtion: ethnol, methnol, ethyl ette, hloroform, etone, n-hexne nd dimethylsulfoxide (DMSO) were purhsed from Merk (Drmstdt, Germny). Folin Ciolteu regent, 2, 2-diphenyl, 1-pirylhydrzine (DPPH) ws quired from Sigm Aldrih (Steinheim, Germny). All other hemils (nlytil grde) nd regents i.e., sodium hydroxide, ferrous hloride, luminum hloride, potssium dihydrogen phosphte, dipotssium hydrogen phosphte, queretin, glli id, sori id, ffei id, rutin, kempferol, (+)-tehin nd myrietin used in this study were purhsed from Merk (Drmstdt, Germny), unless stted otherwise. Preprtion of rude extrt The plnt ws wshed thoroughly under running wter to remove ontmintion nd ws shde dried with tive ventiltion t mient temperture for three weeks. The dried stem, fruit nd leves were ground seprtely to fine powder using eletri knife mill nd stored in irtight ontiners. The powdered plnt prts were sujeted to extrtion y sonition ided mertion using nlytil grde solvents i.e., n-hexne (Nh), hloroform (C), etone (A), ethyl ette + etone (EthA), ethyl ette (Eth), ethnol + hloroform (EC), methnol + hloroform (MC), ethyl ette + ethnol (EthE), methnol + ethyl ette (MEth), methnol (M),

3 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 3 of 18 ethnol (E) nd distilled wter (D). A rtio of 1:1 ws used for prepring extrtion solvent hving vrious solvent omintions s desried ove. Aurtely weighed (40 g) plnt mteril ws merted with 400 ml solvent in 1000 ml Erlenmeyer flsk nd ws sujeted to mertion for 24 h t room temperture followed y sonition (ultrsoni th, room temperture, 30 min). The mr ws extrted twie using sme proedure nd the extrts were omined whih were then filtered through muslin loth followed y filtrtion through Whtmnn No. 1 filter pper. The extrts were then onentrted with vuum evportion in rotry evportor (Buhi, Switzerlnd) nd dried in vuum oven (Ymto, Jpn) t 45 C to otin finl rude extrt nd the experiment ws run in triplite. Perent extrt reovery The dried extrts were weighed to lulte the perent reovery of rude extrt y the following formul. % Extrt reovery ¼ ða=bþ 100 A = Totl weight of dried extrt otined fter drying. B = Totl weight of ground plnt mteril tken for eh extrtion. Phytohemil nlysis Determintion of totl phenoli ontent The totl phenoli ontents were estimted ording to slightly modified proedure s desried previously using Folin Ciolteu regent [5, 6]. An liquot of 20 μl from 4 mg/ml DMSO stok solution of eh test smple ws trnsferred in respetive well of 96 well plte followed y ddition of 90 μl of Folin Ciolteu regent. The plte ws inuted for 5 min fter whih 90 μl of sodium ronte ws dded to the retion mixture. Glli id ws used s stndrd nd sorne of eh retion mixture ws tken fter triplite performne t 630 nm using miroplte reder (Bioteh USA, miroplte reder Elx 800). A lirtion urve (y = x , R 2 = ) ws otined in prllel under the sme operting onditions using glli id ( μg/ml) s positive ontrol nd the orreltion ws found to e signifint t 0.05 level. The ssy ws performed in triplite nd the results re expressed s mg glli id equivlent per grm dry weight (mg GAE/g DW). Determintion of totl flvonoid ontent For totl flvonoid ontent determintion, luminum hloride olorimetri method ws employed with slight modifitions ording to system suitility [7, 8]. The rude extrts (20μl of 4.0 mg/ml DMSO) were trnsferred to eh well of 96 well plte. Susequently, 10 μl eh of 10 % luminum hloride nd 1.0 M potssium ette ws dded followed y the ddition of 160 μl of distilled wter. The resulting mixture ws kept t room temperture for 30 min. Then sorne of the plte ws mesured t 415 nm using miroplte reder. The lirtion urve ws drwn y using queretin s stndrd t finl onentrtions of 0, 2.5, 5, 10, 20, 40 μg/ml, the ssy ws performed in triplite nd the resultnt flvonoid ontent ws doumented in mg equivlents of queretin per grm of plnt dry weight (mg QE/g DW). The eqution otined for the lirtion urve of queretin ws y = x (R 2 = ) nd the orreltion ws found to e signifint t 0.05 level. HPLC-DAD quntittive nlysis High performne liquid hromtogrphy ws performed y using Agilent Chem sttion Rev. B SR1 (260) nd Agilent 1200 series inry grdient pump oupled with diode rry detetor (DAD; Agilent tehnologies, Germny). Reverse phse hromtogrphi nlysis ws rried out with Zorex-C8 nlytil olumn ( mm, 5 μm prtile size, Agilent, USA), injetion volume 20 μl nd the grdient elution ws onduted ording to the method previously desried with minor modifitions [7]. Moile phse onsisted of etonitrile-methnol wter-eti id in rtio of 5:10:85:1 (solvent A) nd etonitrile-methnol-eti id in rtio of 40:60:1(solvent B). Grdient method ws 0 20 min for 0 50 % B, min for % B nd then isorti 100 % B till 30 min. Flow rte ws mintined t 1 ml/min. Stok solutions of vrious phenoli stndrds i.e., phenoli id (glli id), flvn- 3-ol (tehin), flvonol flvonoids (queretin, myrietin, kempferol), hydroxyinnmte (ffei id), flvone glyone (pigenin) nd flvonol glyoside (rutin) were prepred in methnol nd susequently diluted to get finl onentrtion of 10, 20, 50, 100, 200 μg/ml of methnol. The dt for pek re versus stndrd onentrtion ws used to onstrut the lirtion urve, the orreltions were found to e signifint t 0.05 level, results of whih re summrized in Tle 2. The respetive limit of detetion (LOD) nd limit of quntifition (LOQ) s determined y liner regression nlysis of the lirtion urve were lulted y using the expression 3.3 * (σ/) nd 10 * (σ/) respetively where; σ = Stndrd devition of response = Slope of lirtion urve Prior to use, stndrd solutions, smples nd moile phses were ll degssed nd filtered through 0.45 μm memrne filter (Millipore). The sorption of smples ws reorded t 257nm (rutin), 279 nm (glli id, tehin), 325 nm (ffei id, pigenin) nd 368 nm (myrietin, queretin nd kempferol). Chromtogrphi opertion ws rried out t mient temperture nd in triplite. Before strting the

4 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 4 of 18 next nlysis olumn ws reonditioned for 10 min nd the results were expressed s mg/g DW. Comprison of retention time nd UV sorption spetr of extrts with those of stndrds ws done for the identifition of ompounds. Biologil evlution Rdil svenging tivity-dpph ssy The ntioxidnt potentil of the rude extrts ws guged y monitoring its pity to quenh the stle 2, 2-diphenyl 1-pirylhydrzyl (DPPH) free rdil [5, 9]. Spetrophotometri nlysis ws used to mesure the perent rdil svenging pity (%RSA) nd to determine the orresponding 50 % inhiitory (svenging) onentrtion (SC 50 ). The DPPH quenhing ility ws expressed s IC 50, the onentrtion required to inhiit rdil formtion y 50 %. Four different dilutions of eh test extrt (20 μl), to otin finl onentrtions of 200, 66.66, nd μg/ml, were mixed with 180 μl of 9.2 mg/100 ml methnol DPPH solution in 96 well pltes. The sorne ws mesured t 517 nm using miroplte reder fter 30 min of retion t 37 C. Svenging tivity in perent (%RSA) ws lulted y using the eqution: %RSA ¼ ð1 A s =A Þ 100 Where A s is the sorne of DPPH solution with smple, wheres A is the sorne of negtive ontrol ontining the regent exept the smple. Asori id ws used s positive ontrol nd the ssy ws performed in triplite. Totl ntioxidnt pity estimtion y phosphomolydenum sed ssy Phosphomolydenum sed totl ntioxidnt pity of the extrts ws estimted onisely y mixing 0.1 ml of eh test extrt (4 mg/ml DMSO) nd positive ontrol (sori id, 4 mg/ml) with 1 ml of regent (0.6 M sulphuri id, 28 mm sodium phosphte nd 4 mm mmonium molydte). A typil lnk solution onsisted of 1 ml of regent solution nd the pproprite volume of the sme solvent used for eh smple. All tues were inuted in oiling wter th for 90 min t 95 C. After the smples hd een ooled to room temperture, the sorne of the eh smple solution ws mesured t 695 nm ginst the lnk using PDA spetrophotometer (8354 Agilent Tehnologies, Germny). The experiment ws performed in triplite. The ntioxidnt tivity ws expressed s the numer of mg equivlents of sori id per grm of dry plnt weight i.e., mg AAE/g DW [7, 10]. Totl reduing power estimtion y potssium ferriynide olorimetri ssy The reduing power of different solvent extrts ws determined in ordne with the method desried previously [7, 11]. Conisely, 200 μl of eh test extrt (4 mg/ml DMSO) ws mixed with 400 μl of phosphte uffer (0.2 mol/l, ph 6.6) nd 1 % potssium ferriynide [K 3 Fe (CN) 6 ]. The mixture ws then inuted t 50 C for 20 min. A portion of trihloroeti id (400 μl of 10 %) ws dded to the mixture, whih ws then entrifuged t 3000 rpm t room temperture for 10 min. The upper lyer of solution (500 μl) ws mixed with distilled wter (500 μl) nd FeCl 3 (100 μl, 0.1 %). The sorne ws mesured t 700 nm nd n inresed sorne of the retion mixture indited inresed reduing power. Blnk ws prepred y dding 200 μl of DMSO to the forementioned retion mixture insted of the extrt. The reduing power of eh smple ws expressed s mg sori id equivlent per grm plnt dry weight (mg AAE/g DW) nd the ssy ws performed s triplite nlysis. Cytotoxiity ssys: Brine shrimp lethlity ssy A 24 h lethlity test ws performed in 96 well plte ginst rine shrimp (Artemi slin) lrve ording to the previously desried protool with minor modifitions [5]. Eggs of test orgnism Artemi slin (Oen 90, USA) were kept for h hthing period in simulted sterile se wter (38 g/l supplemented with 6 mg/l dried yest) with onstnt oxygen supply in speilly designed two-omprtment plsti try under illumintion, providing diret light nd wrmth (30 32 C). The mture phototropi nuplii were then hrvested with the help of Psteur pipette nd trnsferred to eh well of 96 well plte. Test extrts were initilly tested t three grded onentrtions i.e., 1000, 500 nd 250 μg/ml nd orresponding volume of eh extrt ws then trnsferred to the wells ontining se wter nd shrimp lrve. Positive nd negtive ontrol wells inluded stndrd doxoruiin (4 mg/ml) nd DMSO respetively insted of smple. After 24 h inution period, degree of lethlity exhiited y eh solvent extrt ws determined y ounting the numer of survivors nd medin lethl onentrtion (LC 50 ) of the test smples with 50 % mortlity, ws lulted using tle urve 2D v5.01 softwre. The whole experiment ws run s triplite nlysis. Cytotoxiity ginst THP-1 humn leukemi ell line The in vitro ytotoxiity evlution of extrts ws rried out y using humn leukemi (THP-1) ell lines (ATCC # TIB-202) ording to the previously doumented protool with slight modifition ording to system suitility [5]. Briefly, leukemi ells were ultured in omplete

5 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 5 of 18 growth medium [RPMI-1640 medium uffered with 2.2 g/l NHCO 3 nd supplemented with 10 % het intivted fetl ovine serum (HIFBS); ph 7.4] in ron dioxide inutor (37 C, 5 % CO 2 ). Aout 190 μl of THP-1 ells t seeding density of ells per ml were trnsferred to eh well of 96-well mirotiter plte. Susequently, 10 μl of smple ontining 1 % DMSO in PBS ws dded. Smples were tested three times t onentrtions of 10, 5 nd 2.5 μg/ml. The retion plte ws inuted t 37 C in humidified CO 2 (5 %) inutor for 72 h. Florouril nd vinristine(4mg/mldmso)wereusedspositivestndrd drug in positive ontrol wells wheres 1 % DMSO in PBS served s negtive ontrol. The numer of survivors ws ounted using improved neuuer hmer (Mrien, Germny) under light mirosope nd ompred with perent survivl of ells in the presene of positive nd negtive ontrols nd the ssy ws performed in triplite. Afterwrds LC 50 ws lulted y using tle urve 2D v5.01 softwre. Protein kinse inhiition ssy The protein kinse inhiition ssy ws performed thrie y oserving hyphe formtion in purified isoltes of Streptomyes 85E strin [12]. Bteril lwn ws llowed to develop y spreding spores (myeli frgments) of refreshed ulture of Streptomyes on sterile pltes ontining miniml ISP4 medium. Aout 5 μl of eh extrt (20 mg/ml of DMSO) ws loded onto sterile 6 mm filter pper diss. The impregnted pper diss with finl onentrtion of 100 μg/dis were pplied diretly on the surfe of the pltes seeded with Streptomyes 85E. Surftin nd DMSO infused diss were inluded s positive nd negtive ontrol respetively. The pltes were then inuted t 30 C for 72 h (time required for hyphe formtion in Streptomyes 85E) nd the results were interpreted s ld zone of inhiition round smples nd ontrols infused diss. Antimiroil ssys Antiteril ssy In vitro ntiteril potentil of the given test extrts ws evluted y gr dis diffusion method s desried previously [13]. A lwn of refreshed teril ultures [(grm positive: Stphyloous ureus ATCC-6538, Miroous luteus ATCC-10240) nd grm negtive (Slmonell typhimurium ATCC-14028, Klesiell pneumoni ATCC- 1705)] with pre-djusted seeding density ws mde on nutrient gr pltes. Sterile filter pper diss impregnted with 5 μl (20 mg/ml DMSO) of eh test extrt were pled on the seeded pltes. Dis infused with Cefixime (stndrd ntiteril) served s positive ontrol while DMSO infused dis ws used s negtive ontrol. Following inution t 37 C for 24 h, the verge dimeter of the zone of inhiition round the smple s well s ontrol treted diss ws mesured nd reorded. Extrts produing n inhiition zone 10 mm in dimeter were onsidered tive nd were further sreened to determine minimum inhiitory onentrtions (MICs) y stndrd three-fold miroroth dilution methodology [14]. A stok solution of eh tive extrt ws serilly diluted in 96-well mirotiter plte with Mueller Hinton roth to otin onentrtion rnging from 100 μg/ml to 3.70 μg/ml. A stndrdized inoulum for eh teril strin ws prepred so s to give inoulum size of pproximtely CFU/ml in eh well. Mirotiter pltes were then kept t 37 C for n overnight inution. Following inution, the MIC ws lulted s the lowest onentrtion of the extrt inhiiting the growth of teril strin y mesuring OD t 600 nm nd the ssy ws performed s triplite nlysis. Antifungl ssy The ntifungl potentil of test extrts ws evluted s triplite nlysis y gr dis diffusion method [13]. The spores of given fungl strins [Aspergillus fumigtus (FCBP- 66), Muor speies (FCBP-0300), Aspergillus niger (FCBP-0198) nd Aspergillus flvus (FCBP-0064)] were hrvested in 0.02 % Tween 20 solution nd their turidity ws djusted ording to MFrlnd 0.5 turidity stndrd. Then 100 μl of eh hrvested fungl strin ws swed on pltes ontining Sourud Dextrose gr. Sterile filter pper diss impregnted with 5 μl (20 mg/ml DMSO) eh of test extrt were pled on the seeded pltes. DMSO impregnted dis ws used s negtive ontrol wheres stndrd drug lotrimzole exhiited mximum tivity with 30 ± 1.54 mm zone. Following inution t 28 C for h, the verge dimeter (mm) of the zone of growth inhiition round the smples s well s ontrol treted diss ws mesured nd reorded. Extrts produing n inhiition zone 10 mm in dimeter were sreened to determine minimum inhiitory onentrtions (MICs) t lower onentrtions rnging from 50 to 3.12 μg/dis y stndrd dis diffusion method. MIC ws lulted s the lowest onentrtion of the extrt round whih visile zone of growth inhiition ws formed. Sttistil nlysis The results otined for ytotoxi, ntimiroil nd phytohemil ssys were nlyzed sttistilly y onewy nlysis of vrine (ANOVA) followed y Tukey nd Dunn s test using the sttistil pkge PASW Sttistis 18 nd P < 0.05, P < 0.01, or P < ws onsidered s signifint when pproprite. Dt were expressed s men ± SD. Correltion nlysis of the phytohemil tivities nd HPLC-DAD nlysis ws

6 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 6 of 18 rried out using the orreltion nd regression y Mirosoft Exel progrm. Results nd disussion Effet of extrtion solvent on the extrt yields Perentge of the extrt reovery determined for different plnt prts, using sonition followed y mertion s extrtion tehnique is presented in Tle 1. Mximum mount of extrt ws reovered when distilled wter ws used s the extrtion solvent with n extrt yield of ± 0.87 %, ± 1.18 % nd ± 0.99 % for lef, stem nd fruit respetively. It hs een oserved tht s the polrity of extrtion solvent hnged from highly polr wter to non-polr n-hexne, the extrt yields deresed drstilly. The differenes in the extrt yields in the present nlysis might e endorsed to the different vilility of extrtle omponents, resulting from the vried hemil omposition of plnt metolites. The extrtion effiieny nd iologil tivities re strongly dependent on the nture of extrtion solvent polrity, due to the presene of diverse ompounds of vried hemil hrteristis tht my or my not e solule in prtiulr solvent. As extrtion is the first ritil step in drug disovery proess from plnts, therefore in our present study wide rnge of extrtion solvent polrities nd sonition followed y mertion s extrtion tehnique ws employed. It hs lso een suggested from their extrtion yield Tle 1 Perent extrt reoveries of lef, stem nd fruit of D. innoxi using different extrtion solvents Extrts % Extrt reovery Lef Stem Fruit Nh 0.75 ± ± ± 0.11 C A 6.23 ± ± ± ± 0.22 EthA ± ± 0.62 Eth EC MC EthE ± ± ± ± ± ± ± ± 0.46 MEth 8.55 ± ± 1.96 E M D 8.82 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.94 d ± ± 0.99 Vlues (men ± SD) re verge of three smples of eh plnt prt, nlyzed individully in triplite (n =1 3 3), (P < 0.05); DW dry weight; Supersript letters within the sme olumn indite signifint (P < 0.05) differenes of mens within the extrting solvent; Susript letters within the sme row indite signifint (P < 0.05) differenes of mens within the plnt prts. Nh n-hexne, C Chloroform, A Aetone, EthA Ethyl ette- Aetone, Eth Ethyl ette, EC Ethnol-Chloroform, MC Methnol-Chloroform, EthE Ethyl ette-ethnol, MEth Methnol-Ethyl ette, E Ethnol, M Methnol, D Distilled wter optimiztion studies of herl mteril tht smple preprtion method, mertion under sonition proved to e superltive hoie when onsidering the time/yield rtio [15]. Phytohemil nlysis Totl phenoli ontent The totl phenoli ontent of lef, stem nd fruit of D. innoxi re presented in Fig. 1. In our study the highest ontent of glli id equivlent phenols i.e., ± 0.12 nd ± 0.45 mg/g DW ws present in the queous extrt of lef nd fruit extrts respetively while in se of stem, it ws highest in the ethyl ette-etone extrt (25.06 ± 0.45 mg GAE/g DW). Among lef extrts the phenoli ontent deresed in ordne with the following order of extrtion solvent polrity; D EthA EthE Eth A M E EC MC MEth C Nh. The stem extrts displyed pttern of deresing phenoli ontent in the following order: EthA D Eth MC MEth EthE A EC E C M Nh while it ws D M EthE MC MEth C E EC Eth EthA A Nh in se of fruit extrts. The totl phenols in different plnt prts rnged from ± 0.12 mg GAE/g DW for the highly polr queous to 2.5 ± 0.12 mg GAE/g DW non-polr n-hexne, whih is in greement with the results of previous studies tht quntifition of phenoli ompounds in plnt extrt is influened y the hemil nture of the extrtion solvent (Kumrn et l. [16]). The plnt phenolis hving ntioxidnt properties hve n importnt role in omting oxidtive stress, ytotoxiity nd ell deth y svenging free rdils or helting tre elements therey strengthening the ntioxidnt defenses [17]. In most of the mediinl plnts, phenoli nd polyphenoli ompounds suh s flvonoids, phenoli ids nd tnnins re the mjor ontriutors to the ntioxidnt tivity. Totl flvonoid ontent The totl flvonoid ontent of lef, stem nd fruit in terms of mg queretin equivlent per grm dry weight re presented in Fig. 1. Among ll the lef extrts the highest flvonoid ontent of ± 0.18 mg QE/g DW ws reorded in the ethyl ette-etone extrt followed y D A E Eth M MC = E EC C MEth Nh while in se of stem extrts, the mximum flvonoids were quntified in the ethyl ette extrt yielding out 5.29 ± 1.22 mg QE/g DW. The queous fruit extrt unveiled the highest totl flvonoid ontent of ± mg QE/g DW s ompred to other plnt prts nlyzed. A positive orreltion (orreltion oeffiient; R 2 = for lef, for stem nd for fruit) ws found to e present etween the phenoli nd flvonoid ontents suggesting tht the

7 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 7 of 18 Fig. 1 Lef: TPC (Totl phenoli ontent mg GAE/g DW), TFC (Totl flvonoid ontent mg QE/g DW), TAC (Totl ntioxidnt pity mg AAE/g DW), TRP (Reduing power mg AAE/g DW) nd %RSA (rdil svenging tivity) determintion in different solvent extrts of D. innoxi. Vlues re presented s men ± Stndrd error from triplite investigtion. Nh: n-hexne, C: Chloroform, A: Aetone, EthA: Ethyl ette-aetone, Eth: Ethyl ette, EC: Ethnol-Chloroform, MC: Methnol-Chloroform, EthE: Ethyl ette-ethnol, MEth: Methnol-Ethyl ette, E: Ethnol, M: Methnol, D: Distilled wter. Stem: TPC (Totl phenoli ontent mg GAE/g DW), TFC (Totl flvonoid ontent mg QE/g DW), TAC (Totl ntioxidnt pity mg AAE/g DW), TRP (Reduing power mg AAE/g DW) nd %RSA (rdil svenging tivity) determintion in different solvent extrts of D. innoxi. Vlues re presented s men ± Stndrd error from triplite investigtion. Nh: n-hexne, C: Chloroform, A: Aetone, EthA: Ethyl ette-aetone, Eth: Ethyl ette, EC: Ethnol-Chloroform, MC: Methnol-Chloroform, EthE: Ethyl ette-ethnol, MEth: Methnol-Ethyl ette, E: Ethnol, M: Methnol, D: Distilled wter. Fruit: TPC (Totl phenoli ontent mg GAE/g DW), TFC (Totl flvonoid ontent mg QE/g DW), TAC (Totl ntioxidnt pity mg AAE/g DW), TRP (Reduing power mg AAE/g DW) nd %RSA (rdil svenging tivity) determintion in different solvent extrts of D. innoxi. Vlues re presented s men ± Stndrd error from triplite investigtion. Nh: n-hexne, C: Chloroform, A: Aetone, EthA: Ethyl ette-aetone, Eth: Ethyl ette, EC: Ethnol-Chloroform, MC: Methnol-Chloroform, EthE: Ethyl ette-ethnol, MEth: Methnol-Ethyl ette, E: Ethnol, M: Methnol, D: Distilled wter

8 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 8 of 18 ntioxidnt potentil of phenols might e ttriuted to the presene of flvonoids. HPLC-DAD quntittive nlysis Reverse phse HPLC-DAD sed profiling ws used for quntittive nlysis of seleted plnt phenolis nd the hromtogrphi finger printing ws done y ompring the retention time nd UV spetr of referene ompounds with those of the test smple, the results of whih re summrized in Tles 2, 3 nd 4. A signifint mount of tehin, myrietin, queretin, rutin nd ffei id were quntified in some of the nlyzed extrts. Among the lef extrts sustntil mount of tehin nd pigenin ws present in the methnol (5.41 nd 2.11 μg/mg DW respetively) nd methnolhloroform (1.28 nd 1.78 mg/g DW respetively) extrt. Signifint mount of tehin nd pigenin were lso quntified in ethnoli extrt of fruit (2.65 nd 2.46 mg/g DW respetively). The presene of ll these plnt metolites drw prllel orreltion of plnts potentil with their known iotivities e.g., rutin is one of the phenoli ompounds found in the invsive plnt speies nd ontriutes to its ntiteril nd ntioxidnt properties, ffei id outperformed the other ntioxidnts in reduing fltoxin prodution y more thn 95 % nd pigenin indues utophgy in leukemi ells, whih my support plnt s possile hemopreventive nd ntiner role [18]. The hromtogrms of stndrds s well s ompounds deteted in vrious plnt prts re presented in Fig. 2. Biologil evlution DPPH rdil svenging tivity The perent free rdil svenging tivity (%RSA) of the rude extrts ws ssessed y the disolortion of the methnoli solution of DPPH, the results of whih re summrized in Fig. 1. This method depends on the redution of the purple olored DPPH, y epting eletron or hydrogen rdil from donor ntioxidnt, to stle yellow-olored diphenyl pirylhydrzine moleule (Hq et l. [5]). The est ntioxidnt tivity in DPPH ssy ws demonstrted y the queous extrt of lef (IC 50 = μg/ml) while it ws lowest for n-hexne (IC 50 = μg/ml). Ethyl ette-etone extrt of stem exhiited highest svenging tivity with n IC 50 of μg/ml followed y ethyl ette (IC 50 = μg/ml) nd queous (IC 50 =23.22 μg/ml) extrts. Among ll the fruit extrts the most potent rdil svenging potentil ws exhiited y the queous extrt with n IC 50 of μg/ml. The reltionship etween totl ontents of polyphenols nd the free rdil svenging pity s studied y mny uthors demonstrted tht liner orreltion exists etween them [19]. In our present study signifint orreltion ws lso found etween the rdil svenging pility nd the totl phenoli ontent (lef: R 2 = , stem: R 2 = , fruit: R 2 = ). Therefore it n e inferred tht the vrious polyphenols in D. innoxi extrts re lrgely responsile for the rdil svenging medited ntioxidnt tivity. Totl ntioxidnt pity The totl ntioxidnt pity (TAC) of vrious lef, stem nd fruit extrts re summrized in Fig. 1. The method relies on the redution of Mo (VI) to Mo (V) y the ntioxidnt meditors nd the onsequent formtion of green olored phosphte/mo (V) omplex with mximl sorption t 695 nm (Jfri et l. [7]). Mximum TAC ws displyed y the queous extrt of the lef (46.98 ± 0.14 mg AAE/g DW), ethyl ette extrt of the stem (42.90 ± 1.25 mg AAE/g DW) nd ethnol-hloroform extrt of the fruit (43.86 ± 1.18 mg AAE/g DW). Oxidtion is mndtory nturl phenomenon in iologil systems resulting in the genertion of highly retive hydroxyl nd peroxyl rdil. Antioxidnts quenh these rdils, the indequte endogenous or exogenous supply of whih my use dmge to DNA, proteins nd polyunsturted ftty id residues of ell memrne phospholipids nd my led to pthologil effets suh s vsulr diseses nd ner [20]. The orreltion Tle 2 Clirtion urve prmeters, limit of detetion (LOD), limit of quntifition (LOQ) for the stndrds Stndrd Retention time (min) Clirtion urve eqution Correltion oeffiient (r 2 ) LOD (μg/ml) LOQ (μg/ml) Glli Aid 4.19 y = x Ctehin 7.10 y = x Cffie Aid 9.66 y = x Rutin 3.12 y = x Myrisitin y = x Queritin y = 12.21x Kempherol y = x Apigenin y = x

9 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 9 of 18 Tle 3 Chemil profiling of different solvent extrts of D. innoxi using HPLC-DAD Extrts Polyphenols (μg/mg DW) Phenoli id Flvonol glyoside Hydroxy innmte flvn-3-ol Flvone glyone Flvonol flvonoid GA Rutin CA Cte Api Myr Quer Kemp Lef Nh C A EthA Eth EC MC 1.28 ± ± 0.02 EthE MEth E M 5.41 ± ± ± 0.02 D Stem Nh C A 1.33 ± 0.01 EthA Eth EC MC EthE MEth E 0.18 ± ± 0.03 M D 1.58 ± ± ± ± ± 0.01 Fruit Nh C A 1.75 ± 0.01 EthA Eth EC MC EthE MEth E 2.65 ± ± ± ± 0.01 M D : not deteted, GA glli id, CA ffei id, Cte tehin, Api pigenin, Myr myreetin, Quer queretin, Kemp kempferol

10 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 10 of 18 Tle 4 Retention time (RT) in minutes nd detetion wvelength (λ) in nnometers of deteted ompounds in different solvent extrts of lef, stem nd fruit of D. innoxi Extrts Polyphenols (μg/mg DW) Rutin CA Cte Api Myr Quer Kemp RT (min) λ (nm) RT (min) λ (nm) RT (min) λ (nm) RT (min) λ (nm) RT (min) λ (nm) RT (min) λ (nm) RT (min) λ (nm) Lef MC M Stem A E D Fruit A E : not deteted, GA glli id, CA ffei id, Cte tehin, Api pigenin, Myr myreetin, Quer queretin, Kemp kempferol etween totl phenoli ontent nd ntioxidnt pity ws determined nd it ws found to e liner with n exellent orreltion oeffiient, R 2 of (lef), (stem), (fruit) respetively. These results re in greement with the previous studies whih showed tht high phenoli ontent inreses the ntioxidnt tivity [1, 20]. Reduing power Figure 1 shows the redutive power of vrious solvent extrts of D. innoxi. In our present study, the mximum extrtion effiieny in terms of highest reduing power ws hieved in the queous extrt of lef (9.46 ± 1.12 mg AAE/g DW), ethyl ette extrt of stem (15.35 ± 0.61 mg AAE/g DW) nd methnol-etone extrt (13.90 ± 0.87 mg AAE/g DW) of fruit. The reduing properties re generlly llied with the presene of redutones whih hve een linked to the ntioxidnt tion through rekge of the free rdil hin y donting hydrogen tom, therefore diret orreltion hve een oserved etween the ntioxidnt pity nd reduing power of ertin plnt extrts [20]. In our present explortion positive orreltion ws lso found to exist etween the reduing power nd ntioxidnt potentil of ll the extrts with exellent orreltion oeffiients (R 2 = , , for lef, stem nd fruit respetively) signifint t the P =0.05 level. Cytotoxiity ssys Brine shrimp lethlity ssy Cytotoxiity potentil of the plnt ws tested ginst rine shrimp (Artemi slin) lrve to revel its lethlity profile. Out of totl of 36 orgni extrts sreened for ytotoxi tivity ginst rine shrimp lrve, 25 % of the lef, 16 % of the stem nd 8.3 % of the fruit extrts demonstrted tivity t or elow 100 μg/ml nd were tegorized s highly toxi. The remining 75 % of the lef, 84 % of the stem nd 91.7 % of the fruit extrts hd LC 50 vlues 250 μg/ml nd were tegorized s toxi. The results from sreening of the orgni extrts of different plnt orgns ginst A. slin lrve re shown in Tle 5. The positive ontrol, doxoruiin demonstrted n LC 50 vlue 5.93 μg/ml. Among ll the individul plnt prt extrts, methnol-hloroform ws found to e the most toxi exhiiting n LC 50 of μg/ml for lef nd stem while μg/ml for fruit; inditing tht modertely polr extrtion solvent would e the est hoie for the isoltion of suh ompounds rther thn highly polr or non-polr solvent. The degree of lethlity ws found to e diretly proportionl to the onentrtion of the extrt. The rine shrimp lethlity ssy is well-thought-out s suitle tool for primry evlution of toxiity. It hs lso een proposed for sreening phrmologil tivities in plnt extrts nd the toxiity results n e orrelted with their doumented ethno-phrmologil role. These tests re usully rried to drw extrpoltions on the sfety of the plnt extrts nd further to illustrte trends of their iologil tivities [21]. In iotivity evlution of plnt extrts y rine shrimp iossy, n LC 50 vlue of less thn 1000 μg/ml is onsidered to e ytotoxi. In our urrent study, 100 % of ll the sreened orgni extrts demonstrted LC 50 vlues < 1000 μg/ml, signifying the presene of ytotoxi ompounds responsile for the oserved toxiologil tivity. These results reommended further investigtion of plnt s ytotoxi potentil using in vitro ntiner ell lines.

11 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 11 of 18 Fig. 2 Chromtogrms of stndrd phenols. Chromtogrms of ompounds deteted in () MC: Methnol-Chloroform nd () M: Methnol extrts of D. innoxi lef. Chromtogrms of ompounds deteted in () A: Aetone nd () D: Distilled wter extrts of D. innoxi stem. d Chromtogrms of ompounds deteted in () A: Aetone nd () E: Ethnol extrts of D. innoxi fruit

12 Tle 5 Cytotoxiity nd Streptomyes hyphe formtion inhiition potentil of different solvent extrts of lef, stem nd fruit of D. innoxi. Vlues re presented s men ± stndrd devition of triplite nlysis Extrts Brine shrimp ytotoxiity (Conentrtion μg/ml) THP-1 ytotoxiity (Conentrtion μg/ml) Protein kinse inhiition Lef % mortlity LC 50 % mortlity IC 50 Dimeter (mm ± SD) Cler zone Bld zone Nh ± ± ± ± 0.38 C ± ± ± ± ± ± 0.29 A ± ± ± ± ± 0.47 EthA ± ± ± ± ± 0.39 Eth ± ± ± ± ± 0.47 EC 90.0 ± ± ± ± ± ± 0.52 MC ± ± ± ± ± ± 0.21 EthE ± ± ± ± ± 0.15 MEth ± ± ± ± ± 0.41 E ± ± ± ± ± 0.42 M ± ± ± ± ± 0.35 D ± ± ± Stem Fruit Nh ± ± ± 0.44 C ± ± ± 0.58 A ± ± ± ± 0.42 EthA ± ± ± ± 0.22 Eth ± ± ± ± 0.35 EC ± ± ± ± 0.28 MC ± ± ± ± 0.21 EthE ± ± ± ± 0.45 MEth ± ± ± ± 0.27 E ± ± ± ± 0.33 M ± ± ± ± 0.51 D ± ± ± ± 0.47 Nh ± ± ± ± ± 0.81 C ± ± ± ± ± 0.54 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 12 of 18

13 Tle 5 Cytotoxiity nd Streptomyes hyphe formtion inhiition potentil of different solvent extrts of lef, stem nd fruit of D. innoxi. Vlues re presented s men ± stndrd devition of triplite nlysis (Continued) A ± ± ± ± ± 0.54 EthA ± ± ± ± ± 0.22 Eth ± ± ± ± 0.22 EC ± ± ± ± 0.24 MC ± ± ± ± 0.32 EthE ± ± ± ± ± ± 0.38 MEth ± ± ± ± 0.62 E ± ± ± ± ± 0.67 M ± ± ± ± ± 0.28 D ± ± ± ± 0.56 DMSO Initilly, the smples were evluted t single highest onentrtion nd the smples whih showed more thn 50 % inhiition/signifint tivity were tested t lower onentrtions to find their LC 50. Negtive ontrol: DMSO. LC 50 of Doxoruiin (positive ontrol employed in the rine shrimp lethlity ssy) ws 5.93 μg/ml. IC 50 of 5-Florouril (5FU) nd Vinristine (positive ontrols employed in ntiner ssy) ws 5 nd 8.1 μg/ml respetively. Growth inhiition zone exhiited y surftin (positive ontrol in Protein kinse inhiition ssy) ws 20 ± 1.02 mm (ld zone) Vlues (men ± SD) re verge of three smples of eh plnt extrt, nlyzed individully in triplite (n = 1 3). - Mens differene is highly signifint, slightly signifint, signifint t p < 0.05 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 13 of 18

14 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 14 of 18 Cytotoxiity ginst THP-1 humn leukemi ell line The inidene of severl ners hs inresed exponentilly with ge from the fourth to eighth dede of life. Over 6 million people deese due to ner worldwide eh yer, eing the lrgest single use of deth in oth men nd women [17]. Keeping in view the prodigious ytotoxi potentil s disovered through rine shrimp lethlity ssy; the plnt extrts were further sreened for n in-vitro ytotoxi tivity using humn leukemi (THP-1 ATCC# TIB-202) ell line (Tle 5). Among ll the lef extrts, the hloroform extrt ws most potent s it onsiderly inhiited the ell line prolifertion exhiiting ± 1.77 % ell mortlity t 10 μg/ml onentrtion nd n LC μg/ml. In our present study the stem extrts did not disply ny ntiner potentil while in se of fruit, the most prominent lethlity ws shown y the hloroform nd n-hexne extrts with n LC nd 3.49 μg/ml respetively whih is omprle to the stndrd drugs 5- florouril nd vinristine with 50 % lethlity t 5 μg/ml nd 8.1 μg/ml respetively. Reently new withnolide, dinoxin B ws isolted from methnol extrt of D. innoxi leves exhiited su-miromolr LC 50 vlues ginst multiple humn ner ell lines [4]. This is y fr the first report (to the est of our knowledge) highlighting the ntiner profiieny of D. innoxi fruit phytohemils, whih ording to ll the previous studies hve een reported in its leves only. Protein kinse inhiition ssy The results of protein kinse inhiition zones reorded for the test smples re presented in Tle 5. Among ll the extrts, noteworthy inhiition zone of 22 mm ler, 11 mm ld phenotype ws formed round the ethyl ette extrt of oth lef nd stem, while the most prominent hyphe formtion inhiition in se of fruit ws presented y the ethnol extrt (20 mm ler, 11 mm ld) lgged y etone (18 mm ler, 11 mm ld) nd etone-ethyl ette extrt (17 mm ler, 11 mm ld). The non-toxi effet of DMSO (negtive ontrol) ws onfirmed y the sene of growth inhiition zone wheres surftin, the positive ontrol estlished 16 mm ld growth inhiition zone. The results of the present study suggest tht modertely polr extrtion solvent would e suitle for the extrtion of phytoonstituents of D. innoxi tht my >serve s promising kinse inhiitory trget while the extremes of extrtion solvent polrities exhiited little or no tivity. In the reent yers, there hs een signifint surge for the development of inhiitors of protein kinses from nturl produts espeilly plnts. Protein phosphoryltion t serine/threonine nd tyrosine residues y protein kinses is one of the mjor regultory mehnisms in iologil proesses inluding poptosis, ell prolifertion, ell differentition, nd metolism. Deregulted phosphoryltion t serine/threonine nd tyrosine residues y protein kinses produed s result of geneti ltertions quired erly in tumorigenesis re often the use of ner. In this respet, inhiition of protein kinses hs emerged s promising trget for ner tretment (Yo et l. [12]). Protein kinse tivity is ritil for the eril hyphe formtion of Streptomyes nd this prerequisite hs een exploited in the present study to ioprospet the extrts for their kinse inhiitory tivity so tht their ntiner potentil ould e ssessed. Using Streptomyes 85E s n ssy strin for kinse inhiitors ppers to identify wide rnge of eukryoti kinse modultors, presumly euse the Streptomyete enzymes re evolutionry forerunners of their highly speifi eukryoti ounterprts. An dvntge of the whole ell Streptomyete ssy is tht it redily identifies ytotoxi tivity of the ompounds eing tested. This simple ssy permits the identifition of signl trnsdution inhiitors for vriety of pplitions inluding nti-infetive, ntitumor gents nd severl of the inhiitors of myoteri [22]. Antimiroil ssys Antiteril ssy Preditions of ntiteril tivity in herl ompounds extrted from plnt prts depend lrgely on plnt prt, solvent used for extrtion nd the orgnism tested [14]. Tle 6 shows the ntiteril tivity in terms of zone of inhiition (mm dimeter) of vrious solvent extrts from different prts (leves, stem nd fruit) of D. innoxi. In our study the extrts produing growth inhiitory zone 10 mm in gr dis diffusion ssy were onsidered tive nd were further evluted for MIC determintion through roth miro dilution method. All the extrts showed etter ntiteril tivity ginst Grmpositive teri s ompred to Grm-negtives. Among ll the extrts 39 % of lef, 16 % of stem nd 29 % of fruit extrts were found to e tive (zone 10 mm). Among ll the teril strins tested, Mirooous luteus ws found most suseptile with mximum inhiition y the n-hexne fruit extrt produing zone of inhiition of 24 mm (MIC = 3.70 μg/ml). Dt indited tht extrts prepred from leves possess etter ntiteril tivity thn those prepred from stem nd fruit. Among ll the lef extrts mximum zone of growth inhiition ws displyed y the ethyl ette, ethnol nd etone extrts ginst K. pneumone, S. typhi, M. luteus nd S. ureus respetively. Among ll the stem extrts the mximum growth inhiition zone of 20 mm ws produed y the ethnoli extrt hving MIC of 3.70 μg/ml. The n-hexne fruit extrt presented ntiteril tivity with n inhiition zone rnging etween 7 nd 24 mm. The sene of growth inhiition zones onfirmed the non-toxi effet of DMSO (negtive ontrol) while efixime served s positive ontrol. Hydroxylted phenoli ompounds suh s ffei

15 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 15 of 18 Tle 6 Antiteril tivity of lef, stem nd fruit extrts of D. innoxi Extrts Lef Dimeter of growth inhiition zone (mm ± SD) K. pneumonie MIC (μg/ml) S. typhimurium MIC (μg/ml) M. luteus MIC (μg/ml) S. ureus MIC (μg/ml) Nh 8.5 ± 0.76 d 10 ± ± 1.84 d 9 ± 1.89 C 10 ± ± ± 1.45 d 9 ± 2.31 A 10 ± ± 1.33 d 14.5 ± ± EthA 10 ± ± 1.54 d 12 ± ± 1.78 Eth 12 ± ± ± ± 1.49 d EC 10 ± ± 1.34 d 12 ± ± 1.87 d MC 7 ± 0.45 d 6 ± 2.21 d 16 ± ± 1.89 d EthE 12 ± ± 1.76 d 14 ± ± 2.21 d Meth 9.5 ± ± 1.32 d 10 ± ± 1.38 d E 8 ± 0.67 d 17 ± 1.45 d ± ± 1.22 M 7 ± 0.34 d 8 ± ± ± 1.23 d D 7 ± 0.56 d 7 ± 1.23 d 14 ± ± 1.43 d Stem Fruit Nh 10 ± ± ± C 14 ± ± ± 1.21 A 7 ± 1.21 d 8 ± 2.2 EthA 10 ± ± 1.22 d Eth 7 ± 2.34 d EC 7 ± 1.23 d 13 ± MC 9 ± ± ± 2.21 d EthE 8 ± ± 1.45 d Meth 7 ± 0.77 d E 10 ± ± M 9 ± ± 1.43 d 7 ± 1.23 d 8 ± 1.21 D 10 ± Nh 12 ± ± C 8 ± ± 2.45 d 9 ± ± 1.32 d A 8 ± ± 1.13 d 8 ± 1.45 d EthA 13 ± ± ± EC 8 ± 1.56 d MC 10 ± ± 1.82 EthE 11 ± ± 1.21 d Meth 9 ± ± ± 2.13 d E 10 ± ± ± 1.23 d 12 ± M 7 ± 1.23 d 12 ± ± 1.51 D 10 ± DMSO Cefixime 28 ± ± ± ± Zone of inhiition inluding the dimeter of dis (5 mm). In eh dis, the smple size ws 100 μg per dis (5 μl) in dis diffusion ssy. Vlues (men ± SD) re verge of three smples of eh plnt extrt, nlyzed individully in triplite (n = 1 3). -d Mens differene is highly signifint, slightly signifint, nd signifint t p < : No tivity in dis diffusion ssy or not tive (zone 10 mm) for MIC determintion

16 Ftim et l. BMC Complementry nd Alterntive Mediine (2015) 15:376 Pge 16 of 18 Tle 7 Antifungl tivity of lef, stem nd fruit extrts of D. innoxi tested ginst filmentous fungi Extrts Lef Dimeter of growth inhiition zone (mm ± SD) A. fumigtus MIC μg/ml Muor sp. MIC μg/ml A. niger MIC μg/ml A. flvus MIC μg/ml Nh 8 ± ± ± 2.21 d 100 C 8 ± ± 1.32 d 100 A 7 ± 1.56 d ± ± EthA 7 ± 2.45 d ± ± 1.78 Eth 7 ± 1.67 d 100 EC MC EthE 7 ± 1.22 d ± Meth 9 ± E 7 ± 2.43 d ± M 9 ± ± 1.67 d 100 D 8 ± Stem Fruit Nh 10 ± ± ± C 14 ± ± ± A 7 ± 1.21 d ± EthA 10 ± ± 1.22 d 100 Eth 7 ± 2.34 d 100 EC 7 ± 1.23 d ± MC 9 ± ± ± 2.21 d 100 EthE 8 ± 2.14 d ± 1.45 d 100 MEth 7 ± 0.77 d 100 E 10 ± ± 1.82 d 100 M 9 ± ± 1.43 d ± 1.23 d ± D 10 ± Nh 12 ± ± C 8 ± 1.23 d ± 2.45 d ± ± A 8 ± 1.45 d ± 1.13 d ± 1.45 d 100 EthA 13 ± ± ± Eth 16 ± EC 8 ± 1.56 d 100 MC 10 ± ± EthE 11 ± ± 1.21 d 100 MEth 9 ± ± ± 2.13 d 100 E 10 ± ± ± 1.23 d ± M 7 ± 1.23 d ± ± D 10 ± Zone of inhiition inluding the dimeter of dis (5 mm). In eh dis, the smple size ws 100 μg per dis (5 μl) in dis diffusion ssy. Vlues (men ± SD) re verge of three smples of eh plnt extrt, nlyzed individully in triplite (n = 1 3). -d Mens differene is highly signifint, slightly signifint, nd signifint t p < : No tivity in dis diffusion ssy or not tive (zone 10 mm) for MIC determintion