Antioxidant, Antimicrobial, Cytotoxic, and Protein Kinase Inhibition ...downloads.hindawi.com/journals/bmri/2019/6478187.pdf · ResearchArticle Antioxidant, Antimicrobial, Cytotoxic,

Research ArticleAntioxidant Antimicrobial Cytotoxic and ProteinKinase Inhibition Potential in Aloe vera L

Huma Tariq1 Muhammad Zia 2 Ihsan-ul-Haq3

Syed AunMuhammad4 Shujaat Ali Khan1 Nighat Fatima1 Abdul Mannan 1

ArshadMehmood Abbasi 5 andMingxing Zhang 6

1Department of Pharmacy COMSATS University Islamabad (Abbottabad Campus) Pakistan2Department of Biotechnology Quaid-i-Azam University Islamabad 45320 Pakistan3Department of Pharmacy Quaid-i-Azam University Islamabad 45320 Pakistan4Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan5Department of Environment Sciences COMSATS University Islamabad (Abbottabad Campus) Pakistan6The First Affiliated HospitalSchool of Clinical Medicines Guandong Pharmaceutical University Guangzhou 510006 China

Correspondence should be addressed to Abdul Mannan amannanciitnetpkArshad Mehmood Abbasi arshad799yahoocom and Mingxing Zhang zmingxing126com

Received 5 February 2019 Revised 17 June 2019 Accepted 20 June 2019 Published 28 July 2019

Academic Editor Yun-Peng Chao

Copyright copy 2019 Huma Tariq 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

Aloe vera is amultifunctional plant that has gained acceptance as an excellent home remedy source inAsia and theworldThepresentstudy was intended to evaluate the phytochemical contents and in vitro antioxidant antimicrobial antileishmanial and proteinkinase inhibition activities in different fractions of A vera leaf Methanolic extract of A vera leaves was fractionated using columnchromatography and ten fractions (AV1-AV10) were obtained Phenolics composition antioxidant antimicrobial antileishmanialand protein kinase inhibition activities were evaluated using standard protocols Well-known compounds of A vera were used forin silico study against enzymes involved in brine shrimp and antileishmanial and hyphae formation inhibition assay on the basisof results Five fractions (AV3 to AV7) possess potential total phenolics and flavonoids contents along with significant biologicalactivities AV4 fraction exhibited the highest total phenolics content 3324 plusmn 326120583g GAEmg and total antioxidant activity 1504plusmn 25815120583g AAEmg determined by phosphomolybdenum complex assay Fraction AV6 showed 95 antileishmanial effect as wellas the lowest LD

50value of 05305120583gmL in brine shrimp lethality assay The Protein Kinase inhibition potential in A vera leaves

was determined for the first time and three fractions AV1 AV6 and AV7 depicted activity with the highest zone of inhibition upto 21plusmn05mm (AV7) Docking analysis showed that A vera contains anthraquinones anthrones chromones and polysaccharidesresponsible for synergistic cytotoxic antileishmanial antibacterial and antioxidant potential of this plant Therefore with morestudies A vera could probably have the potential to be used for drug development against leishmaniasis

1 IntroductionThehistory of development ofmedicinal components is basedon the fact that over the centuries the natural products such astaxol artemisinin andmorphine were used to cure a numberof diseases [1] The natural products are more acceptablethan purely synthetic products because they match biologicalintermediates and endogenous substances and have suitabil-ity in active transport mechanisms [2] Nowadays plantsbased secondary metabolites extracted from crude extract orfractions are good source of diverse chemical structures that

show potent biological profile and pharmacological activities[3 4]

Thousands of polyphenolics compounds such as phe-nolics and flavonoids have been discovered so far Thesecompounds retain different antioxidant antibacterial anti-cancerous antiviral and anti-inflammatory activities andantiatherosclerotic properties [5 6] The pharmacologicaland physiological potentials of phenolic compounds dependon their free radical scavenging and antioxidant activities andproperties tomaintain the activity of enzymes responsible for

HindawiBioMed Research InternationalVolume 2019 Article ID 6478187 14 pageshttpsdoiorg10115520196478187

2 BioMed Research International

detoxification [7] A very unique health effect of flavonoidsis that they are cardioprotective due to inhibition of LDLoxidation [8 9] The ability of flavonoids to transfer stablefree radicals and metal catalyst chelation provides protectionto the biological systems [10] antioxidant enzymes activation[11] alpha-tocopherol radical reduction [12] and inhibitionof enzyme oxidases [13]

Globally infectious diseases are considered major causeofmortality andmorbidity each yearThe infectious disordersare caused by organisms such as bacteria viruses fungi orparasites [14 15] Among bacterial infections certain bacteriaare well known such as S aureus E coli and PseudomonasRecently most of bacterial strains become resistant to exist-ing antibacterial compounds and pose major problem fortreatment [16] Candida albicans is considered the mostcommonly occurring fungus responsible for infections [17]In case of protozole infections leishmaniasis and malaria aremain health problems around the world particularly in thedeveloping countries [18]

Therefore it is essential to screen various plants for theirantimicrobial antiprotozoal potential [14 19] Antimicrobialassays include agar well diffusion disc diffusion and brothdilution methods Infections related to cancer also needattention due to emergence of resistance Cytotoxicity andenzyme inhibitory studies can play an important role indrug development against cancer [20] The brine shrimplethality bioassay is important and effective assay to screencytotoxic potential while various enzymes inhibition assaysfor example protein kinase inhibition aromatase inhibitionand iNOS inhibition assays are used for anticancer studies[21] Protein kinases show a huge class of enzymes thatplay an important role in regulation of complex molecularmechanisms that control various functions of the cellsincluding survival proliferation and apoptosis Many patho-logic states appear due to abnormal protein kinase functionsincluding cancer inflammatory and autoimmune disordersand cardiac diseases Now the protein kinases are one of themajor therapeutic targets [22] For the treatment of cancerProtein kinase inhibitors are a well-known family of clinicallyeffective drugs especially in treating cancer [23]

The genusAloe belonging to family Liliaceae exists almostall over the world and is extensively disseminated in theAfrican and the eastern European continents The genusAloe has more than 400 species including globally tradedA vera Aloe ferox and Aloe arborescens [24] Aloe genusis reported for many medicinal uses such as its use intreating constipation gastrointestinal disorders and immunesystem deficiencies Aloe vera also showed pharmacologicalactivities including antioxidant antimicrobial antitumorhypoglycemic hypolipidemic and antidiabetic ones [25]These properties are mainly contributed by inner gel of theleaves and presence of more than 200 different biologicallyactive substances [24] The medicinal and pharmacologicalpotential of A vera revealed that it is quite auspicious as aversatile therapeutic plant and should be further investigatedIn this context the present study was designed to evaluatetotal phenolics content in vitro antioxidant and antimicrobialproperties cytotoxicity and protein kinase inhibition activityin various fractions of Aloe vera leaf extract Some of the

well-known secondarymetabolites ofA vera plant belongingto anthraquinone resins anthracenes and polysaccharideclass were used for in silico study against enzymes involved inhyphae formation of Streptomyces hatching ofArtemia salinalarvae and growth of leishmanial parasite

2 Materials and Methods

21 Sampling The plant samples were collected fromHaripur which lies between 34∘-341015840 and 34∘ndash161015840 Northlatitude and 77∘-331015840 and 73∘-211015840 East longitude KhyberPakhtunkhwa Pakistan September 2014 and is identifiedby taxonomist at Quaid-i-Azam University Islamabad Plantsamples were collected from private land by the verbalpermission of the land owner to conduct this study About13 kg fresh leaves were cleaned with tap water followedby distilled water and then chopped in the grinder andshade-dried with continuous agitation for 2 weeks

22 Extraction Approximately 388g dried plantmaterial wasmacerated in 16 liters of methanol and kept for ten days atroom temperature with continuous stirring and shaking atleast 4 to 5 times daily The primary separation was donewith muslin cloth followed by filtration using Whatmanfilter paper Filtrate was dried and evaporated under reducedpressure at 40∘C in a rotary evaporator and crude extract(20g) was stored for further analysis

23 Fractionation by Column Chromatography The crudeextract was fractionated by using column chromatography[26] After TLC optimization methanol and chloroform wereselected for column chromatography Concentrated solutionof crude extract was prepared in selected solvents and loadedon blank silica gel (1g x 15g) to prepare sample for columnchromatography

Silica gel (60 70-230 mesh MERCK) was used as sta-tionary phase and the variation of solvent combinations ofincreasing polarity was used as mobile phase The bottom ofthe glass columnwas stocked with cotton pad and slurry (600g of silica gel and 600mL of chloroform)was carefully pouredinto the column with continuous tapping Then previouslyprepared dry power of extract and silica gel was poured onthe top of the column and column packing was completedby putting blank silica on the top Solvent was poured fromthe top and opened the tap of the column Elution of theextract was done with 400 mL of each solvent combination(methanol Chloroform) that is 0100 298 397 595 7931090 1387 1684 1981 2377 2971 3565 4060 5050 6040and 7030 vv In total 55 elution fractions were collected in100mL glass bottlesThe fractions present in each bottle werestudied by TLCmethod tomix the closely related fractions onthe basis of spots observed inUV light Fractions that showedthe same spots were pooled together and total ten fractionswere obtained that is AV1 AV2 AV3 AV4 AV5 AV6 AV7AV8 AV9 and AV10

24 Estimation of Total Phenolics Total phenolics content(TPC) in different fractions of A vera leaf (AV1-AV10)was determined following the method of [27] with slight

BioMed Research International 3

modification In brief 20 120583L of each fraction was taken in96-well plate followed by addition of 90 120583L diluted Folin-Ciocalteu reagent in each well The mixture was incubatedfor 5 min at room temperature and 90 120583L of 6 sodiumcarbonate solution was added and again incubated for 1 hourat room temperature The absorbance was measured at 630nm wavelength on microplate reader Final value of totalphenolics content was expressed as Gallic acid equivalent(GAE) and data were expressed as plusmn SD for triplicate analysis

25 Estimation of Total Flavonoids Total flavonoids content(TFC) was estimated by ammonium chloride calorimetricmethod as described by [28] Briefly 20 120583L of each fractionin triplicate was taken in 96-well plate followed by addition of10 120583L of potassium acetateThen 10 120583L of aluminum chloride(10) was added to the reaction mixture and constituted upto 200 120583L using distilled water Then mixture was incubatedfor 30 minutes at 25∘C Quercetin was used as positivecontrol and DMSO was used as negative control or blankAbsorbance was measured at 405 nm on microplate readerThe TFC value was expressed as Quercetin equivalent anddata were presented as plusmn SD for triplicate analysis

26 Assessment of Antioxidant Activity Antioxidant activityin different fractions ofA vera leaf was determined using dif-ferent assays such as 2 2-diphenyl-1-picrylhydrazyl (DPPH)ferric ion reducing power assay and phosphomolybdenumcomplex assay

261 DPPH Assay The DPPH free radical scavengingassay is established to evaluate the scavenging potentials ofantioxidants against the stable 2 2-diphenyl-1-picrylhydrazyl(DPPH) radical For the estimation of antioxidant scav-enging activity DPPH assay is thought to be an authenticand simple assay [29] The DPPH free radical scavengingactivity of A vera leaf fractions was measured by followingthe method of [27] Briefly 10 120583L of the analyte solutionwas added in 96-well plates followed by addition of 190120583L of DPPH testing agent Ascorbic acid (1mgml) waspositive control while DMSO was used as negative controlAbsorbance was measured at 517 nm on microplate readerafter one hour of incubation (37∘C) The percentage DPPHradical scavenging activity was calculated using the formula

DPPH scavenging = (1 minus AsAc) lowast 100 (1)

where As is the absorbance of sample and Ac is theabsorbance of control Data were expressed as plusmn SD fortriplicate analysis

262 Ferric Ion Reducing Power Assay Reducing power ispowerful impression of antioxidant activity of a compound[30] Compounds that have to reduce may interact withpotassium ferricyanide (Fe3+) and reduce it to potassium fer-rocyanide (Fe2+) which subsequently interacts with ferrouschloride in order to generate complex of ferric ferrous whichat 700nm wavelength shows maximum absorption [31] Theferric ion reducing power was estimated by following themethod as described earlier [32] Briefly 200 120583L of each

fraction was taken in Eppendorf tube followed by theaddition of 400120583L of 02MPhosphate buffer (pH66) and 500120583L of 1Potassium ferricyanideThenmixturewas incubatedfor 20 min at 50∘C Then 400 120583L of 10 Trichloroacetic acidsolution was mixed and centrifuged at 3000 rpm for 10 minThen 150 120583L of supernatant was added in 96-well plates Afterthat 50 120583L of 01 of ferric chloride was mixed to each wellcontaining sample mixture The 200 120583L of ascorbic acid (1mgmL) was used as positive control and equal volume ofDMSO was used as negative control The absorbance wastaken at 630 nm on microplate reader and results of ferricion reducing power were presented as AA equivalent 120583gmLData were expressed as plusmn SD for triplicate analysis

263 Phosphomolybdenum Complex Assay The total antiox-idant potential is also accessed through generation of com-plex compound called phosphomolybdenum In this assaytheMo (VI) is reduced toMo (V) through the test sample andfurther generation of Mo(V) green phosphate complex at apH which is acidic [33] Total antioxidant potential of AV1-AV10 fractions was determined by phosphomolybdenumcomplex assay as explained previously [32] In short 100120583L ofeach fractionwas taken in 96-well plate and 900 120583L of reagentsolution [4 mM ammonium molybdate 28 mM sodiumphosphate 06 M sulfuric acid 163 mL conc sulfuric acid16795g NaH

2SO4 and 0247g ammoniummolybdate and 50

mL distilled water] was added The test blend was incubatedat 95∘C for 90 min Ascorbic acid (1 mgmL) solution waspositive control while DMSO was used as negative controlThe reading was taken at 630 nm wavelength on microplatereader and results of total antioxidant activity were presentedas AA equivalent 120583gmL Data were expressed as plusmn SD fortriplicate analysis

27 Antibacterial Assay Antibacterial activity of A veraleaf fractions against gram positive Staphylococcus aureus(ATCC6538) andMicrococcus luteus (ATCC 10240) and gramnegative Escherichia coli (ATCC 15224) and Pseudomonasaeruginosa was determined by microtiter plate method [34]The bacterial culture was formulated in nutrient broth andincubated for 12 hrs and then stored in refrigerator at 4∘CBefore testing culture was transferred to incubator for 14 hrsand diluted 10 folds with nutrient broth (110) 5 120583L of thesample solution (4 mgmL sample solution + 20 DMSOand nutrient broth) was taken in 96-well plate Then 195 120583Lof freshly diluted inoculums were added to each well andthe final test concentration was 100120583gmLThe active samplewas tested at lower concentration by 3 timesrsquo serial dilutionthat is 333 111 and 37120583gmL The Cefixime monohydratewas used as positive control at 10 333 111 and 037 120583gmLwhile 5120583L of 20 DMSO in nutrient broth was taken asnegative control Absorbance was measured at 630 nm after24 hrs incubation through microplate reader and percentageinhibition was calculated

28 Antifungal Assay Four fungal strains Mucor speciesAspergillus flavus Fusarium solani and Aspergillus nigerwere used to test the antifungal potential in studied samplesusing disc diffusion as explained earlier [35] The fungal


strains were stored on SDA at 4∘C Terbinafine (4mgmLin DMSO) and pure DMSO were used as positive andnegative control respectively Terbinafine disc concentrationwas 20 120583g Four growing of fungal strains were cultured inSabouraud dextrose agar (SDA) having 65 pH 5 120583L of eachcrude extract solution was spew on the surface of filter paperdiscs and were implanted on the surface of the media in thePetri plate The concentration of the sample and control was100120583gdisc The prepared Petri dishes were incubated for 72hours at 28∘C and after incubation the inhibition zones weremeasured

29 Antileishmanial Activity The in vitro antileishmanialactivity of Aloe fractions was determined by using themethod as described before [36] with slight adjustmentsTheleishmanial strain (Leishmania tropicaKWH23) was culturedin medium 199 containing 10 heat-inactivated Fetal BovineSerummaintained at 24 plusmn 1∘C for 6-7 daysThe amphotericinB was used as positive control drug and DMSO was used asnegative control In 96-well plates the stock solutions (4 mgof crude fraction + 1000 120583L of DMSO) were diluted seriallyTheDMSOwas used as negative control and Amphotericin Bwas used as positive control The 96-well plate was incubatedfor 72 hours at 24∘C After the incubation 15 120583L of assayculture was transferred to counting chamber (Neubauer) forlive promastigotes counting using light microscope Tablecurve 2D version 4 was used for LC

50calculation Data were

expressed as plusmn SD for triplicate analysis

210 Cytotoxicity Assay The cytotoxic potential in A veraleaf fractions was estimated through brine shrimp lethalitytest as discussed earlier [37] with some modifications Atfirst stock solution of samples was formulated (100mg crudefractionmL DMSO) then 500 250 100 and 10 120583gmLdilutionswere formulated Different dilutions ofDoxorubicin(1mg of doxorubicin + 1 mL DMSO) that is 10 1 and 01120583gmL were used as standard drug A shallow rectangulartray (22x32 cm) already was filled with simulated sea water(38g sea saltL of distilled water) in which brine shrimpeggs (Artemia salina L purchased from Sara HeidelbergGermany) hatched The tray was consisting of two portionsone of which was large and the other was small Each portionwas separated from the other through a wall consisting ofmany holes In the large portion of the tray brine shrimprsquoseggs were spread on the surface of simulated sea water Afterspreading the brine shrimprsquos eggs the large portion of thetraywas concealedwith aluminum foilThe surface of smallerportion was lighted up with a lamp and hatching started after24-26 hoursThe newly brine shrimp larvae (nauplii) traveledtowards the light portion These nauplii were taken from thetank and shifted to the beaker by Pasteur pipette

50 120583L of sample solution with four concentrations (500250 100 and 10120583g) was poured into respective well of 96-well plate followed by addition of 200 120583L of the simulatedsea water and mixed carefully Ten shrimps were countedunder a 3x magnifying glass and then transferred to eachwell with the help of Pasteur pipette and 300 120583L volumewas maintained for each well to accomplish the requiredconcentration of crude sample The microplates containing

shrimps were incubated at room temperature for 24 hrsThenshrimpswere taken out from thewell with Pasteur pipette andsurvivor ratewas counted under a 3xmagnifying glasswith 3xresolution against a background that is lighted The values ofLD50(sample concentration required to kill 50 of shrimps)

were calculated by using Table curve 2D version 4

211 Protein Kinase Inhibition Assay Theprotein kinase inhi-bition potential was studied through a method establishedon the restriction of hyphae formulation in Streptomyces asexplained before [38 39]The sample solutionwas formulatedby mixing 4 mg fractions1000120583L DMSO In 20 mL oftryptic soya broth culture was grown for 3 to 4 days withshaking at 30∘C The production of hyphae inhibition teston Streptomyces was carried out The Streptomyces myceliafragments were dispersed on the surface of ISP4 agar mediaplates Then 5 120583L of the test sample was applied to each diskand placed carefully on the agar surface previously seededwith Streptomyces Two types of zones were appeared thatis clear and bald type around the area of paper disk whichweremeasured after 30 to 48 hrs of incubationThe inhibitionzone greater than 9 mm revealed that the sample is activeThe Surfactin and DMSO were used as positive control andnegative control respectively

212 Molecular Docking Chemical structures of compounds1-10 as mentioned in Table 1 were selected from NCBIdatabase and drawn by ChemBioDraw The legends wereconverted into MOL format for further use The 3D crystalstructure of serine protease (UniProt ID A8D853) (PDBID 2HLC) trypanothione reductase (PDB ID 2W0H) andtyrosinase (PDB ID 3NM8) enzymes involved in hatchingof Artemia larvae growth of Leishmania and formation ofStreptomyces respectively was accessed and downloadedfrom Protein Data Bank (PDB) database [httpwwwrcsborgpdbhomehomedo] The active sites of target proteinswere analyzed using the Molecular Operating Environ-ment (MOE) software [httpswwwchemcompcomMOE-Molecular Operating Environmenthtm] An active site wasdefined from the coordinates of the ligand in the originaltarget protein sites

A computational ligand-target docking approach wasused to determine structural complexes of receptor targetswith ligands molecules in order to understand the structuralbasis of these proteins targets specificity Finally dockingwas carried out byMolecularOperating Environment (MOE)software The energy of interaction of these compounds withthe protein targets is assigned ldquogrid pointrdquo [39]

3 Statistical Analysis

All tests relevant to composition and properties assessmentof A vera were performed in triplicate Data were presentedas mean plusmn SD from at least three replicates

4 Results and Discussion

41 Total Phenolics and Flavonoids Contents The measuredvalues of TPC and TFC in different fractions of A vera


Table 1 Details of compounds used for in silico analysis

S Code Name of compound1 Compound 1 Aloin (anthraquinone glycoside)2 Compound 2 Aloe emodin (anthraquinone)3 Compound 3 Mannan (polysaccharide)4 Compound 4 Isoaloresin D (chromones)5 Compound 5 Emodin (hydroxyl anthraquinone)6 Compound 6 Aloe barbendol7 Compound 7 Physcion (anthraquinone derivative)8 Compound 8 Aloetic acid9 Compound 9 Cinnamic acid10 Compound 10 Galactan

Table 2 TPClowast and TFClowast contents in different fractions of A vera

Fractions TPC (120583g GAE eqmg) TFC (120583g QE eqmg)AV1 1397 plusmn 151 5187 plusmn 120AV2 1362 plusmn 133 5128 plusmn 789AV3 1803 plusmn 219 8754 plusmn 155AV4 3324 plusmn 326 6047 plusmn 137AV5 3046 plusmn 296 5103 plusmn 987AV6 1578 plusmn 154 3247 plusmn 657AV7 1197 plusmn 133 1846 plusmn 768AV8 1044 plusmn 120 1949 plusmn 579AV9 1133 plusmn 195 2267 plusmn 460AV10 9795 plusmn 215 2487 plusmn 312TPClowast total phenolics TFClowast total flavonoids

(AV1 to AV10) are given in Table 2 AV4 fraction exhibitedthe highest level of total phenolics content (3324 plusmn 326 120583gGAEmg) followed by AV5 AV3 and AV6 (3046 plusmn 2961803 plusmn 219 and 1578 plusmn 154 120583g GAEmg resp) The lowestvalue of total phenolics content was showed by AV10 (9795plusmn 215 120583g GAE eqmg) These findings are in agreementwith each other as reported previously [40 41] It has beenreported that plants with high phenolic contents have woundhealing ability and reduce inflammation However methodof extraction has a great impact on the value of total phenoliccontent so more phenolics were determined in ethanol andchloroform extracts compared to water extract [42 43]

The anti-inflammatory and antimicrobial potential ofplants is attributed to flavonoids content In the studiedfractions TFC content ranged from 1846 plusmn 768 to 8754plusmn 155120583g QEmg AV3 fraction exhibited the highest TFCwhereas the lowest value was calculated for AV7In A verafractions measured levels of TFC were comparatively lowerthan those reported previously [42] Furthermore type ofsolvent may also contribute in the extract amount so moreTFC content was reported in methanol-chloroform extractthan aqueous extract [35 41]

42 Antioxidant Potential DPPH radical scavenging ferricion reducing potential and total antioxidant activity assayswere conducted to determine the antioxidant potential inmethanolic fractions of A vera leaf The percentage of DPPH

radical scavenging capacity of various fractions as shown inFigure 1(a) ranged from 1182 to 7554 AV5 fraction has thehighest DPPH scavenging activity (7554 plusmn 116) followedby AV6 and AV4 (5787 plusmn 653 and 5299 plusmn 901 resp)These values were comparable to previously reported levelsin the ethanolic extract of A vera leaf [42] however theywere a little lower than those reported earlier [41] Howevermore DPPH radical scavenging activity has been reported inthe methanolic and ethanolic extracts ofA vera leaf and pulpthan n-hexane and aqueous extracts [44 45]

Results of ferric ion reducing antioxidant power (FRAP)are shown in Figure 1(b) All tested fractions showed signifi-cant ferric ion reducing ability however AV5 fractions exhib-ited the highest potential 2289 plusmn 391120583g AA eqmg to reduceferric ion followed by AV6 (2086 plusmn 293 120583g AA eqmg) andAV4 (1959 plusmn 312 120583g AA eqmg) However AV10 fractionexhibited the lowest FRAP value 3618 plusmn 796120583g AA eqmgIn a study on leaf skin of A vera ethanol-chloroform extractdepicted the highest reducing power activity compared toethyl acetate butanol and n-hexane extract [46] Thereforevariation in FRAP values might be due to difference inplant parts used harvesting time geoclimatic conditions andsolvent system used for the extraction as reported [47] Theresults of total antioxidant capacity (TAC) determined byphosphomolybdenum complex assay are given in Figure 2The total antioxidant activity in AV1 to AV10 A vera leaffractions ranged from 2877 plusmn 936 to 1504 plusmn 258120583g AA


1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

DPP

H ac

tivity

(a)

1 2 3 4 5 6 7 8 90 1110

A era fractions

0

50

100

150

200

250

300

Ferr

ic io

n re

duci

ng p

ower

(g

AA

eqm

g)

(b)

Figure 1 Free radicals scavenging potential in different fractions of A vera leaf (a) DPPH activity (b) Ferric ion reducing antioxidantpotential

1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

120

140

160

180

200

Tota

l ant

ioxi

dant

activ

ity (

g A

A eq

mg)

Figure 2 Total antioxidant activity of A vera leaf fractions

eqmg with the highest TAC value in AV4 and the lowest inAV8 fraction Three fractions AV4 AV3 and AV5 exhibitedthe highest TAC These findings are in compliance to [46]

43 Antimicrobial Potential The antibacterial potential ofstudied samples given in Table 3 revealed that crude fractionswere not much effective against E coli but relatively goodresults were obtained for P aeruginosa The most significantinhibition was shown by AV8 AV6 AV10 and AV9 fractionsagainst P aeruginosa with MIC values of 070 071 772 and868120583gmL respectively The AV4 AV7 and AV9 fractionsexhibited good response againstM luteuswithMIC values of103 161 and 250120583gmL respectively In the previous studiesregarding A vera DMSO gel extract ethanolic extract wasfound active against E coli P aeruginosa and S aureus [48ndash50] However antibacterial potential of A vera fractions inthe present evaluation against E coli was not in supportwith reported data However Lawrence et al [50] reportedsignificant potential in methanolic extract of A vera againstE coli which is partially in agreement with the current study

140

120

100

80

60

40

20

0

Mor

talit

y (

)

1 2 3 4 5 6 7 8 9 10

DMSO

Amphoterici

n B

A era fractions and control

Figure 3 Antileishmanial activity of A vera leaf fractions

The antifungal activity of A vera fractions against fourstrains that is Aspergillus niger Mucor specie Aspergillusflavus and Fusarium solani is mentioned in Table 4 Allthe tested fractions exhibited minor effects against fungalstrains which is not in complete agreement with previousreports [51 52] This variation of biological potential may bedue to solvent use harvesting time seasonal variation andecological factors

The antileishmanial activity carried out at 50 120583gmLconcentration in different fractions ofA vera leaf extract andresults are given in Figure 3 The highest value of mortalitywas shown by AV6 fraction (95) followed by AV7 AV9and AV4 (92 84 and 83 resp) whereas the lowest valuewas shown by AV10 (18) The results were comparable withprevious studies [53ndash55]

44 Cytotoxicity of A vera Fractions Thebrine shrimp lethal-ity assay is considered as the most appropriate assay for the


Table3Antibacteria

lactivity

ofA

vera

leafrsquosfractio

ns

Fractio

nsMinim

uminhibitory

concentration(M

IC)

Mluteus

Saureus

Ecoli

Paeruginosa

inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)AV

15793

325

6915

250

5595

673

7600

6020

AV2

4961

1000

6026

500

5702

874

7026

1860

AV3

6314

500

7376

250

4843

1000

8025

1002

AV4

6340

103

6744

lt125

4380

gt1000

7710

6466

AV5

4961

1000

6675

lt125

5119

971

7710

1239

AV6

4807

1000

6915

lt125

4449

gt1000

6924

0710

AV7

5457

161

6769

250

4555

1000

7042

1250

AV8

4878

1000

6504

125

4217

gt1000

7671

0700

AV9

5226

250

6274

125

4724

1000

7482

8670

AV10

5335

1000

7060

250

4154

100

7404

7720

Cefixime

8693

058

1002

101

5119

058

8693

0200


Table 4 Antifungal activity of A vera leaf rsquos fractions

Fractions Zone of inhibition (mm)A niger Mucor spp A flavus F solani

AV1 --- 8319 plusmn 12 7198 plusmn 07 ---AV2 --- --- 6264 plusmn 13 7245 plusmn 19AV3 --- 9017plusmn 15 8451 plusmn 16 9364 plusmn 20AV4 --- 1025 plusmn 13 6137 plusmn 07 8170 plusmn 13AV5 --- 7352 plusmn 09 9326 plusmn 15 1003 plusmn 06AV6 --- 8036 plusmn 07 7367 plusmn 08 9217 plusmn 15AV7 --- 8247 plusmn 13 - - - 8612 plusmn 08AV8 --- 9364 plusmn 17 8353 plusmn 17 ---AV9 --- --- 8632 plusmn 20 8219 plusmn 05AV10 --- 1115 plusmn 20 --- ----ve control (DMSO) --- --- --- ---+ve control (Terbinafine ) 2215 plusmn 19 2437 plusmn 25 2747 plusmn 19 2519 plusmn 27

Table 5 Cytotoxic activity of Aloe vera leaf fractions

Fractions Mortality ()500 120583gmL 250 120583gmL 100 120583gmL 10 120583gmL LD

50(120583gmL)

AV1 67 62 58 38 3505AV2 69 60 56 41 4972AV3 80 67 60 50 9070AV4 80 70 62 54 4710AV5 80 72 63 52 4710AV6 75 69 64 62 0530AV7 73 55 54 47 6413AV8 50 50 42 37 4128AV9 40 30 0 0 gt 500AV10 60 55 50 35 1076Doxorubicin 100 94 89 70 0270

pharmacological active crude plant extract whichmay exhibitlethality against newly hatched nauplii [56] In the presentstudy cytotoxicity of A vera leaf fractions was determinedby Brine Shrimp Lethality assay and results in LD

50(120583gmL)

value are mentioned in Table 5 The fractions with low LD50

value are more active and have high cytotoxic potentialTherefore AV6 fraction depicted the highest cytotoxicitywith LD

50value of 0530120583gmL followed by AV4 AV5

and AV3 (4710 4710 and 9070 120583gmL resp) A studycarried out on cytotoxicity of A vera was reported againstbrine shrimp and LD

50was estimated over 500120583gmL [20]

which is partially correlated with the current study Howeverbrine shrimp lethality activity in methanolic extract ofMeliaazedarach stem bark with327120583gmL LD

50value [57] is in

agreement with our findings

45 Protein Kinase Inhibitions Figure 4 depicted proteinkinase inhibition (PKI) activity in themethanolic fractions ofA vera leaf At 4mgmL concentration only three fractionsAV7 AV6 and AV2 showed PKI activity The AV7 fractionexhibited the highest zone of inhibition (21 plusmn 058mm)followed by AV2 and AV6 (12 plusmn 045 and 95 plusmn 037mm

30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)

AV1 AV2 AV3 AV4 AV5 AV6 AV7 AV8 AV9AV1

0

DMSO

Surfa

ctin

A era fractions and controls

Figure 4 Protein Kinase inhibitions in A vera leaf fraction

resp)The results are strongly supported by a study of Yao andcoworkers which was carried out on evaluation of hyphaeformation inhibition in Streptomyces 85E and isolated com-pounds showed 21mm zone of inhibition at 80120583gdisk and itwas hypothesized that the compounds prevent the formation


(a) (b)

(c)

Figure 5 Potential isolated binding sites of each target protein analyzed by Molecular operator Environment (MOE) software (a) Serineprotease (b) Trypanothione reductase (c) Tyrosinase

of hyphae in Streptomyces 85E which may inhibit cancerproliferation [38]

Comparatively measured levels of secondary metabolitesand biological activities particularly antioxidant potential indifferent fractions of A vera were less than those in previousreports Such variations may be attributed to diverse factorsincluding genetic variation in A vera reported from variousregions harvesting time and storage conditions differencein geoclimatic conditions and growing environment such astemperature precipitation humidity altitude salinity anddrought stress along with the analytical techniques used

46 Molecular Docking Study

461 Isolation of Binding Sites Potential isolated bindingsites of each target protein were analyzed by Molecularoperator Environment (MOE) software The possiblebinding sites residues of serine protease (UniProt IDA8D853) (PDB ID 2HLC) were as follows ILE17 TRP41GLN73 TYR74 TRP141 GLY142 GLN143 SER144 ASN145THR146 ASP147 THR152 VAL153 ILE154 GLN156CYS191 PHE192 GLY193 GLY217A ALA218 GLY219CYS220 GLU221 and SER222 (Figure 5(a)) Similarly in caseof trypanothione reductase (PDB ID 2W0H) binding siteresidues were LEU10 GLY11 ALA12 GLY13 SER14 GLY15GLY16 VAL34 ASP35 VAL34 ASP35 VAL36 ALA46ALA47 GLY49 GLY50 THR51 CYS52 VAL55 GLY56CYS57 LYS60 GLY125 PHE126 GLY127 GLU141 ALA159

THR160 GLY161 SER162 TRP165 ALA200 GLU202PHE203 GLY229 PHE230 ASP231 LEU283 ALA284ILE285 GLY286 ARG287 VAL288 PRO289 ARG290SER291 GLN292 GLN292 ALA293 LEU294 ASN306ILE325 GLY326 ASP327 VAL328 ASN330 ARG331VAL332 MET233 LEU334 THR335 PRO336 and ALA338(Figure 5(b)) Binding site residues of tyrosinase (PDBID 3NM8) were ASP36 ILE39 ALA40 TRP41 GLY43ALA44 LYS47 PHE48 HIS49 ILE139 ASP140 GLU141GLN142 GLY143 PRO219 THR220 ASN223 and TYR267(Figure 5(c)) Protein geometric arrangement of amino acidresidues in allowed and disallowed regions indicates thequality of target proteins

462 Docking Analysis Molecular docking was performedto find ideal ligand orientation with protein for stablecomplex formation Strength of association between ligandand protein is evaluated on the basis of scoring functionand minimum binding energy Computational ligand-targetbinding approachwas used in analyzing structural complexesof compounds with three selected enzymes in order tointerpret structural basis of target protein specificity Theinteraction energy of compounds with target enzyme isassigned ldquogrid pointrdquo Finally these ligandswere dockedwiththe potential active sites of target molecules Compounds 3(mannan) 4 (isoaloresin D) and 9 (cinnamic acid) showedgood results against serine protease with binding energyabove -50 (Table 6Figures 6(a) and 6(b)) while rest of the


Table 6 Energy values obtained during docking analysis of compounds as ligand molecules serine protease (PDB ID 2HLC) enzyme astarget molecules

S no mol rseq mseq S Rmsd-refine E-conf E-place E-score1 E-refine E-score21 COMPD1 2HLC 1 1 -4522 4681 1683 -1105 -2026 -1283 -45222 COMPD2 2HLC 1 2 -3696 4430 3388 -1261 -1864 -9617 -36963 COMPD3 2HLC 1 3 -5279 4460 3384 -1260 -2634 -1328 -52794 COMPD4 2HLC 1 4 -5249 3720 1462 -1212 -1625 -9643 -52495 COMPD5 2HLC 1 5 -3651 3198 3287 -4726 -1716 -6049 -36516 COMPD6 2HLC 1 6 -4149 3672 4029 -9750 -1675 -1155 -41497 COMPD7 2HLC 1 7 -4204 3778 4710 -1051 -1984 -1138 -42048 COMPD8 2HLC 1 8 -3785 2240 -1762 -5366 -1126 -8136 -37859 COMPD9 2HLC 1 9 -5034 4061 2816 -1166 -2248 -1640 -503410 COMPD10 2HLC 1 10 -3932 3282 2261 -4539 -1820 -7129 -3932

(a)

(b)

Figure 6 Views of molecular docking and information of ligands interaction with atoms of serine protease target performed by MolecularOperating Environment (MOE) software comp-4 (a) and comp-9 (b) as mentioned in Table 1

compounds also showed binding potential These finding arealso correlated with the fact that A vera extract showed cyto-toxic activities against cell lines [58] In case of effects of thesecompounds when analyzed against trypanothione reductaseenzyme responsible for leishmanial parasitersquos growth it wasobserved that compounds 3 (mannan) 5 (emodin) and 6(aloe barbendol) showed the lowest binding energy (Table 7)and against tyrosinase compound 8 (aloetic acid) showedbetter results while compounds 2 (aloe emodin) 3(mannan)4 (isoaloresin D) and 9 (cinnamic acid) also showed someactivity indicating their potential for Streptomyces inhibition(Table 8Figures 7(a) and 7(b)) These findings are corre-lated with previous reports that Aloe vera plant contains

anthraquinones anthrones chromones and polysaccharidesresponsible for anticancer antileishmanial antibacterial andantioxidant potential of plant [53 59] Docking results indi-cating the lowest binding energy cluster were considered asrepresentative binding statesTheminimumbinding energiesshowed that target proteins were docked successfully withligand molecules

5 Conclusion

Thepresent studywas focused to assess in vitro protein kinaseinhibition brine shrimp lethality assay and antioxidant activ-ity of different factions (AV1 to AV10) of A vera leaf extract


Table 7 Energy values obtained during docking analysis of compounds as ligand molecules trypanothione reductase (PDB ID 2W0H)enzyme as target molecules

S no mol rseq mseq S Rmsd-refine E-conf E-place E-score1 E-refine E-score21 COMPD1 2W0H 1 1 -5950 6927 1543 -1406 -1574 -3376 -59502 COMPD2 2W0H 1 2 -5943 4344 3324 -1284 -1506 -1751 -59433 COMPD3 2W0H 1 3 -7136 3591 3699 -1929 -1705 1333 -71364 COMPD4 2W0H 1 4 -1220 2907 1951 -1497 -1359 7064 -12205 COMPD5 2W0H 1 5 -6221 2175 3906 -1034 -1325 -8982 -62216 COMPD6 2W0H 1 6 -6025 3343 3880 -1154 -1522 -1599 -60257 COMPD7 2W0H 1 7 -5581 2251 6232 -1335 -1486 2153 -55818 COMPD8 2W0H 1 8 -5148 3238 -1671 -7216 -994 -1981 -51489 COMPD9 2W0H 1 9 -2591 3356 3438 -1829 -1586 5147 -259110 COMPD10 2W0H 1 10 -5958 2460 2519 -1118 -1474 -1042 -5958

Table 8 Energy values obtained during docking analysis of compounds as ligand molecules tyrosinase (PDB ID 3NM8) enzyme as targetmolecules

S no mol rseq mseq S Rmsd-refine E-conf E-place E-score1 E-refine E-score21 COMPD1 3NM8 1 1 7123 2412 3187 -1311 -1968 1265 71232 COMPD2 3NM8 1 2 -4240 3577 4918 -1312 -1590 1381 -42413 COMPD3 3NM8 1 3 -4964 6082 3382 -1532 -2394 -1451 -49654 COMPD4 3NM8 1 4 -4963 5343 1246 -8528 -1432 -1557 -49635 COMPD5 3NM8 1 5 -2949 3719 5649 -9654 -1563 2546 -29496 COMPD6 3NM8 1 6 -2244 1677 5600 -9459 -1635 3625 -22457 COMPD7 3NM8 1 7 1947 2255 1605 -1353 -1875 8287 19488 COMPD8 3NM8 1 8 -5016 1550 -1619 -6369 -1049 -1049 -50179 COMPD9 3NM8 1 9 -4328 3061 3062 -1383 -1944 -9012 -432810 COMPD10 3NM8 1 10 -3495 2725 2707 -9846 -1734 2328 -3495

(a)

(b)

Figure 7 Views of molecular docking and information of ligands interaction with atoms of tyrosinase target performed by MolecularOperating Environment (MOE) software comp-3 (a) and comp-8 (b) as mentioned in Table 2


The AV4 AV5 AV6 and AV7 methanolic fractions depictedsignificant TPC and TFC contents and antioxidant andantimicrobial activities along with cytotoxicity and proteinkinase inhibition The Protein Kinase Inhibitory assay wasperformed first time and AV7 depicted the highest valueof 21 plusmn 050mm Our findings revealed that A vera is aversatile medicinal plant with significant biological activitiesand could be used for future drug against cancer andleishmaniasis

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

All authors declared that they have no conflicts of interest

Authorsrsquo Contributions

Huma Tariq performed research experiments Muham-mad Zia and Ihsan-ul-Haq were involved in data analysisSyed Aun Muhammad and Nighat Fatima contributed inreagentsmaterials and data interpretation Abdul Mannansupervised the project ArshadMehmoodAbbasi contributedin data compilation analysis and write-up Mingxing Zhangcontributed in final draft editing and funding

Acknowledgments

Research facilities provided by COMSATS University Islam-abad (Abbottabad Campus) are gratefully acknowledgedwhereas financial and technical aid provided byDrMingxingZhang to complete this project is thankfully acknowledged

References

[1] G M Rishton ldquoNatural products as a robust source of newdrugs and drug leads past successes and present day issuesrdquoAmerican Journal of Cardiology vol 101 no 10 pp S43ndashS492008

[2] A Ganesan ldquoThe impact of natural products upon moderndrug discoveryrdquo Current Opinion in Chemical Biology vol 12no 3 pp 306ndash317 2008

[3] X Han T Shen and H Lou ldquoDietary polyphenols andtheir biological significancerdquo International Journal of MolecularSciences vol 8 no 9 pp 950ndash988 2007

[4] R W Owen A Giacosa W E Hull R Haubner B Spiegel-halder andH Bartsch ldquoThe antioxidantanticancer potential ofphenolic compounds isolated from olive oilrdquo European Journalof Cancer vol 36 no 10 pp 1235ndash1247 2000

[5] D J Newman G M Cragg and K M Snader ldquoNaturalproducts as sources of new drugs over the period 1981ndash2002rdquoJournal of Natural Products vol 66 no 7 pp 1022ndash1037 2003

[6] A V Krishnaraju T V Rao D Sundararaju M Vanisree H STsay and G V Subbaraju ldquoAssessment of bioactivity of Indianmedicinal plants using brine shrimp (Artemia salina) lethality

assayrdquo International Journal of Applied Science and Engineeringvol 3 no 2 pp 125ndash134 2005

[7] A G Baidez P Gomez J A Del Rıo and A Ortuno ldquo Dys-functionality of the Xylem inOlea europaea L Plants Associatedwith the Infection Process by Verticillium dahliae Kleb Role ofPhenolic Compounds in Plant Defense Mechanism rdquo Journal ofAgricultural and Food Chemistry vol 55 no 9 pp 3373ndash33772007

[8] J B Harborne and C A Williams ldquoAdvances in flavonoidresearch since 1992rdquo Phytochemistry vol 55 no 6 pp 481ndash5042000

[9] A Mazur D Bayle C Lab E Rock and Y RayssiguierldquoInhibitory effect of procyanidin-rich extracts on LDL oxida-tion in vitrordquo Atherosclerosis vol 145 no 2 pp 421-422 1999

[10] M Ferrali C Signorini B Caciotti et al ldquoProtection againstoxidative damage of erythrocyte membrane by the flavonoidquercetin and its relation to iron chelating activityrdquo FEBSLetters vol 416 no 2 pp 123ndash129 1997

[11] A J Elliott S A Scheiber C Thomas and R S PardinildquoInhibition of glutathione reductase by flavonoids A structure-activity studyrdquo Biochemical Pharmacology vol 44 no 8 pp1603ndash1608 1992

[12] R Hirano W Sasamoto A Matsumoto H Itakura O Igarashiand K Kondo ldquoAntioxidant ability of various flavonoids againstDPPH radicals and LDL oxidationrdquo Journal of NutritionalScience and Vitaminology vol 47 no 5 pp 357ndash362 2001

[13] P Cos L Ying M Calomme et al ldquoStructure-activity relation-ship and classification of flavonoids as inhibitors of xanthineoxidase and superoxide scavengersrdquo Journal of Natural Prod-ucts vol 61 no 1 pp 71ndash76 1998

[14] M L Colombo and E Bosisio ldquoPharmacological activitiesof Chelidonium majus L (papaveraceae)rdquo PharmacologicalResearch vol 33 no 2 pp 127ndash134 1996

[15] D M Morens G K Folkers and A S Fauci ldquoThe challengeof emerging and re-emerging infectious diseasesrdquo Nature vol430 no 6996 pp 242ndash249 2004

[16] J E Bandow H Brotz L I Leichert H Labischinski andM Hecker ldquoProteomic approach to understanding antibioticactionrdquo Antimicrobial Agents and Chemotherapy vol 47 no 3pp 948ndash955 2003

[17] M A Kabir M A Hussain and Z Ahmad ldquoCandida albicansa model organism for studying fungal pathogensrdquo InternationalScholarly Research Notices vol 12 pp 1ndash15 2012

[18] L Rocha J Almedia R Macedo and J Barbosa-Folho ldquoAreview of natural products with antileishmanial activityrdquo Phy-tomed vol 12 pp 514ndash535 2005

[19] M Iwu A R Duncan and C O Okunji New Antimicrobialsof Plant Origin Perspectives on new crops and new uses ASHSPress Alexandria Egypt 1999

[20] K Thu Y Y Mon T A Khaing and O M Tun ldquoStudy onphytochemical properties antibacterial activity and cytotoxi-city of aloe vera lrdquo International Journal of Biotechnology andBioengineering vol 77 pp 102ndash106 2013

[21] N Fatima T P Kondratyuk E-J Park et al ldquoEndophyticfungi associated with Taxus fauna (West Himalayan Yew) ofPakistan potential bioresources for cancer chemopreventiveagentsrdquo Pharmaceutical Biology vol 54 no 11 pp 2547ndash25542016

[22] G Scapin ldquoProtein Kinase Inhibition Different Approaches toSelective Inhibitor Designrdquo Current Drug Targets vol 7 no 11pp 1443ndash1454 2006


[23] L A Smyth and I Collins ldquoMeasuring and interpreting theselectivity of protein kinase inhibitorsrdquo Journal of ChemicalBiology vol 2 no 3 pp 131ndash151 2009

[24] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditiola and Complementry Medicines vol5 no 1 pp 21ndash26 2014

[25] M D Boudreau P W Mellick G R Olson R P Felton B TThorn andFA Beland ldquoClear evidence of carcinogenic activityby a whole-leaf extract of Aloe barbadensis Miller (Aloe vera)in F344N ratsrdquo Toxicological Sciences vol 131 no 1 pp 26ndash392013

[26] O Ode I Asuza and I Ajayi ldquoBioassay-guided fractionation ofthe crude methanol extract of cassia singueana leavesrdquo Journalof Advanced Scientific Research vol 2 2011

[27] G Clarke K Ting C Wiart and J Fry ldquoHigh correlationof 22-diphenyl-1-picrylhydrazyl (DPPH) radical scavengingferric reducing activity potential and total phenolics contentindicates redundancy in use of all three assays to screen forantioxidant activity of extracts of plants from the malaysianrainforestrdquo Antioxidants vol 2 no 1 pp 1ndash10 2013

[28] C C Chang M H Yang H M Wen and J C ChernldquoEstimation of total flavonoid content in propolis by twocomplementary colorimetric methodsrdquo J Food Drug Anal vol10 2002

[29] L M McCune and T Johns ldquoAntioxidant activity in medicinalplants associated with the symptoms of diabetes mellitus usedby the Indigenous Peoples of theNorth American boreal forestrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 197ndash2052002

[30] M Oktay I Gulcin and O I Kufrevioglu ldquoDetermination ofin vitro antioxidant activity of fennel (Foeniculum vulgare) seedextractsrdquo LWT - Food Science and Technology vol 36 no 2 pp263ndash271 2003

[31] P Jayanthi and P Lalitha ldquoReducing power of the solventextracts of Eichhornia crassipes (Mart) Solmsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 3 no 3pp 126ndash128 2011

[32] R Ullah I Hussain and S Ahmad ldquoPhytochemical andBiological Evaluation of Phlomis bracteosa A Reviewrdquo LifeScience Journal vol 10 2013

[33] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[34] I Wiegand K Hilpert and R E W Hancock ldquoAgar and brothdilution methods to determine the minimal inhibitory concen-tration (MIC) of antimicrobial substancesrdquo Nature Protocolsvol 3 no 2 pp 163ndash175 2008

[35] N Akhtar and B Mirza ldquoPhytochemical analysis and compre-hensive evaluation of antimicrobial and antioxidant propertiesof 61 medicinal plant speciesrdquo Arabian Journal of Chemistry2015

[36] I Khan M M Yasinzai Z Mehmood I Ilahi and J KhanldquoComparative study of green fruit extract of melia azedarachlinn with its ripe fruit extract for antileishmanial larvicidalantioxidant and cytotoxic activityrdquo American Journal of Phy-tomedicine and Clinical Therapeutics vol 2 pp 442ndash454 2014

[37] A S Apu S H Bhuyan F Khatun M S Liza and MMatin ldquoAssessment of cytotoxic activity of twomedicinal plants

using brine shrimp (Artemia salina) as an experimental toolrdquoInternational Journal of Pharmaceutical Sciences and Researchvol 4 pp 1125ndash1130 2013

[38] G Yao F M Sebisubi L Y C Voo C C Ho G T Tan and LC Chang ldquoCitrinin derivatives from the soil filamentous fungusPenicillium spH9318rdquo Journal of the BrazilianChemical Societyvol 22 no 6 pp 1125ndash1129 2011

[39] S A Muhammad S Ahmed A Ali et al ldquoPrioritizing drugtargets in Clostridium botulinumwith a computational systemsbiology approachrdquo Genomics vol 104 no 1 pp 24ndash35 2014

[40] M Kammoun S Miladi Y B Ali M Damak Y Gargouriand S Bezzine ldquoIn vitro study of the PLA2 inhibition andantioxidant activities of Aloe vera leaf skin extractsrdquo Lipids inHealth and Disease vol 10 article no 30 2011

[41] A Siahpoosh and S Dehdari ldquoPolyphenolic contents andantioxidant activities of leaves of phoenix dactylifera andflowers of aloe verardquo Intetnational Journal of Biolosciences vol5 pp 294ndash304 2014

[42] M Moniruzzaman B Rokeya S Ahmed A Bhowmik M IKhalil and S H Gan ldquoIn vitro antioxidant effects of aloe bar-badensis miller extracts and the potential role of these extractsas antidiabetic and antilipidemic agents on streptozotocin-induced type 2 diabetic model ratsrdquo Molecules vol 17 no 11pp 12851ndash12867 2012

[43] S Petti and C Scully ldquoPolyphenols oral health and disease areviewrdquo Journal of Dentistry vol 37 no 6 pp 413ndash423 2009

[44] QHu Y Hu and J Xu ldquoFree radical-scavenging activity ofAloevera (Aloe barbadensis Miller) extracts by supercritical carbondioxide extractionrdquo Food Chemistry vol 91 no 1 pp 85ndash902005

[45] V Saritha ldquoAntioxidant and antibacterial activity of Aloevera gel extractsrdquo International Journal of Pharmaceutical andBiological Archive vol 1 2010

[46] S Miladi and M Damak ldquoIn vitro antioxidant activities of aloevera leaf skin extractsrdquo J de la Societe Chimique de Tunisie vol10 pp 101ndash109 2008

[47] S W Qader M A Abdulla L S Chua N Najim M MZain and S Hamdan ldquoAntioxidant total phenolic content andcytotoxicity evaluation of selectedMalaysian plantsrdquoMoleculesvol 16 no 4 pp 3433ndash3443 2011

[48] J Antonisamy N Beaulah R Laju and G Anupriya ldquoAnti-Bacterial and Antifungal activity of Aloe Vera gel extractrdquoInternational Journal of Biomedical and Advance Research vol3 no 3 pp 184ndash187 2012

[49] R Lawrence P Tripathi and E Jeyakumar ldquoIsolation Purifi-cation and Evaluation of Antibacterial Agents from Aloe verardquoBrazilian Journal of Microbiology vol 40 no 4 pp 906ndash9152009

[50] M Stanley O Ifenyi and O Eziokwu ldquoAntimicrobial effects ofAloe vera on some human pathogensrdquo International Journal ofCurrentMicrobiology andApplied Sciences vol 3 pp 1022ndash10282014

[51] T A Khaing ldquoEvaluation of the antifungal and antioxidantactivities of the leaf extract of aloe vera (aloe barbadensismillerrdquo Proceedings of World Academy of Science Engineeringand Technology vol 75 pp 610ndash612 2011

[52] U Sitara N Hassan and J Naseem ldquoAntifungal activity of Aloevera gel against plant pathogenic fungirdquo Pakistan Journal ofBotany vol 43 no 4 pp 2231ndash2233 2011

[53] A Dutta G Mandal C Mandal and M Chatterjee ldquoIn vitroantileishmanial activity of Aloe vera leaf exudate a potential


herbal therapy in leishmaniasisrdquoGlycoconjugate Journal vol 24no 1 pp 81ndash86 2007

[54] A Dalimi M Delavari F Ghaffarifar and J Sadraei ldquoInvitro and in vivo antileishmanial effects of aloe-emodin onLeishmania majorrdquo Journal of Traditional and ComplementaryMedicine vol 5 no 2 pp 96ndash99 2015

[55] A C D Queiroz T D L F Dias C B B D Matta L H AC Silva and J X D Araujo-Junior ldquoAntileishmanial activity ofmedicinal plants used in endemic areas in northeastern brazilrdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 478290 9 pages 2014

[56] B N Meyer N R Ferrigni J E Putnam L B Jacobsen DE Nichols and J L McLaughlin ldquoBrine shrimp a convenientgeneral bioassay for active plant constituentsrdquo Planta Medicavol 45 no 5 pp 31ndash34 1982

[57] M Shalabi K Khilo M M Zakaria M G Elsebaei WAbdo and W Awadin ldquoAnticancer activity of Aloe vera andCalligonum comosum extracts separetely on hepatocellularcarcinoma cellsrdquo Asian Pacific Journal of Tropical Biomedicinevol 5 no 5 pp 375ndash381 2015

[58] S Chen K Lin C Chang C Fang and C Lin ldquoAloe-emodin-induced apoptosis in human gastric carcinoma cellsrdquo Food andChemical Toxicology vol 45 no 11 pp 2296ndash2303 2007

[59] S Husain MA Alam N Jahan S Ahmed and H SKauserSibr ldquoAloe vera and its therapeutic efficacy described inUnani Medicine a reviewrdquo Journal of Scientific and InnovativeResearch vol 3 no 5 pp 545ndash551 2014

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detoxification [7] A very unique health effect of flavonoidsis that they are cardioprotective due to inhibition of LDLoxidation [8 9] The ability of flavonoids to transfer stablefree radicals and metal catalyst chelation provides protectionto the biological systems [10] antioxidant enzymes activation[11] alpha-tocopherol radical reduction [12] and inhibitionof enzyme oxidases [13]

Globally infectious diseases are considered major causeofmortality andmorbidity each yearThe infectious disordersare caused by organisms such as bacteria viruses fungi orparasites [14 15] Among bacterial infections certain bacteriaare well known such as S aureus E coli and PseudomonasRecently most of bacterial strains become resistant to exist-ing antibacterial compounds and pose major problem fortreatment [16] Candida albicans is considered the mostcommonly occurring fungus responsible for infections [17]In case of protozole infections leishmaniasis and malaria aremain health problems around the world particularly in thedeveloping countries [18]

Therefore it is essential to screen various plants for theirantimicrobial antiprotozoal potential [14 19] Antimicrobialassays include agar well diffusion disc diffusion and brothdilution methods Infections related to cancer also needattention due to emergence of resistance Cytotoxicity andenzyme inhibitory studies can play an important role indrug development against cancer [20] The brine shrimplethality bioassay is important and effective assay to screencytotoxic potential while various enzymes inhibition assaysfor example protein kinase inhibition aromatase inhibitionand iNOS inhibition assays are used for anticancer studies[21] Protein kinases show a huge class of enzymes thatplay an important role in regulation of complex molecularmechanisms that control various functions of the cellsincluding survival proliferation and apoptosis Many patho-logic states appear due to abnormal protein kinase functionsincluding cancer inflammatory and autoimmune disordersand cardiac diseases Now the protein kinases are one of themajor therapeutic targets [22] For the treatment of cancerProtein kinase inhibitors are a well-known family of clinicallyeffective drugs especially in treating cancer [23]

The genusAloe belonging to family Liliaceae exists almostall over the world and is extensively disseminated in theAfrican and the eastern European continents The genusAloe has more than 400 species including globally tradedA vera Aloe ferox and Aloe arborescens [24] Aloe genusis reported for many medicinal uses such as its use intreating constipation gastrointestinal disorders and immunesystem deficiencies Aloe vera also showed pharmacologicalactivities including antioxidant antimicrobial antitumorhypoglycemic hypolipidemic and antidiabetic ones [25]These properties are mainly contributed by inner gel of theleaves and presence of more than 200 different biologicallyactive substances [24] The medicinal and pharmacologicalpotential of A vera revealed that it is quite auspicious as aversatile therapeutic plant and should be further investigatedIn this context the present study was designed to evaluatetotal phenolics content in vitro antioxidant and antimicrobialproperties cytotoxicity and protein kinase inhibition activityin various fractions of Aloe vera leaf extract Some of the

well-known secondarymetabolites ofA vera plant belongingto anthraquinone resins anthracenes and polysaccharideclass were used for in silico study against enzymes involved inhyphae formation of Streptomyces hatching ofArtemia salinalarvae and growth of leishmanial parasite

2 Materials and Methods

21 Sampling The plant samples were collected fromHaripur which lies between 34∘-341015840 and 34∘ndash161015840 Northlatitude and 77∘-331015840 and 73∘-211015840 East longitude KhyberPakhtunkhwa Pakistan September 2014 and is identifiedby taxonomist at Quaid-i-Azam University Islamabad Plantsamples were collected from private land by the verbalpermission of the land owner to conduct this study About13 kg fresh leaves were cleaned with tap water followedby distilled water and then chopped in the grinder andshade-dried with continuous agitation for 2 weeks

22 Extraction Approximately 388g dried plantmaterial wasmacerated in 16 liters of methanol and kept for ten days atroom temperature with continuous stirring and shaking atleast 4 to 5 times daily The primary separation was donewith muslin cloth followed by filtration using Whatmanfilter paper Filtrate was dried and evaporated under reducedpressure at 40∘C in a rotary evaporator and crude extract(20g) was stored for further analysis

23 Fractionation by Column Chromatography The crudeextract was fractionated by using column chromatography[26] After TLC optimization methanol and chloroform wereselected for column chromatography Concentrated solutionof crude extract was prepared in selected solvents and loadedon blank silica gel (1g x 15g) to prepare sample for columnchromatography

Silica gel (60 70-230 mesh MERCK) was used as sta-tionary phase and the variation of solvent combinations ofincreasing polarity was used as mobile phase The bottom ofthe glass columnwas stocked with cotton pad and slurry (600g of silica gel and 600mL of chloroform)was carefully pouredinto the column with continuous tapping Then previouslyprepared dry power of extract and silica gel was poured onthe top of the column and column packing was completedby putting blank silica on the top Solvent was poured fromthe top and opened the tap of the column Elution of theextract was done with 400 mL of each solvent combination(methanol Chloroform) that is 0100 298 397 595 7931090 1387 1684 1981 2377 2971 3565 4060 5050 6040and 7030 vv In total 55 elution fractions were collected in100mL glass bottlesThe fractions present in each bottle werestudied by TLCmethod tomix the closely related fractions onthe basis of spots observed inUV light Fractions that showedthe same spots were pooled together and total ten fractionswere obtained that is AV1 AV2 AV3 AV4 AV5 AV6 AV7AV8 AV9 and AV10

24 Estimation of Total Phenolics Total phenolics content(TPC) in different fractions of A vera leaf (AV1-AV10)was determined following the method of [27] with slight










































1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

DPP

H ac

tivity

(a)

1 2 3 4 5 6 7 8 90 1110

A era fractions

0

50

100

150

200

250

300

Ferr

ic io

n re

duci

ng p

ower

(g

AA

eqm

g)

(b)


1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

120

140

160

180

200

Tota

l ant

ioxi

dant

activ

ity (

g A

A eq

mg)




140

120

100

80

60

40

20

0

Mor

talit

y (

)

1 2 3 4 5 6 7 8 9 10

DMSO

Amphoterici

n B







Table3Antibacteria

lactivity

ofA

vera

leafrsquosfractio

ns

Fractio

nsMinim

uminhibitory

concentration(M

IC)

Mluteus

Saureus

Ecoli

Paeruginosa

inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)AV

15793

325

6915

250

5595

673

7600

6020

AV2

4961

1000

6026

500

5702

874

7026

1860

AV3

6314

500

7376

250

4843

1000

8025

1002

AV4

6340

103

6744

lt125

4380

gt1000

7710

6466

AV5

4961

1000

6675

lt125

5119

971

7710

1239

AV6

4807

1000

6915

lt125

4449

gt1000

6924

0710

AV7

5457

161

6769

250

4555

1000

7042

1250

AV8

4878

1000

6504

125

4217

gt1000

7671

0700

AV9

5226

250

6274

125

4724

1000

7482

8670

AV10

5335

1000

7060

250

4154

100

7404

7720

Cefixime

8693

058

1002

101

5119

058

8693

0200







50(120583gmL)



50(120583gmL)







50value [57] is in



30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)


0

DMSO

Surfa

ctin





(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of











































1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

DPP

H ac

tivity

(a)

1 2 3 4 5 6 7 8 90 1110

A era fractions

0

50

100

150

200

250

300

Ferr

ic io

n re

duci

ng p

ower

(g

AA

eqm

g)

(b)


1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

120

140

160

180

200

Tota

l ant

ioxi

dant

activ

ity (

g A

A eq

mg)




140

120

100

80

60

40

20

0

Mor

talit

y (

)

1 2 3 4 5 6 7 8 9 10

DMSO

Amphoterici

n B







Table3Antibacteria

lactivity

ofA

vera

leafrsquosfractio

ns

Fractio

nsMinim

uminhibitory

concentration(M

IC)

Mluteus

Saureus

Ecoli

Paeruginosa

inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)AV

15793

325

6915

250

5595

673

7600

6020

AV2

4961

1000

6026

500

5702

874

7026

1860

AV3

6314

500

7376

250

4843

1000

8025

1002

AV4

6340

103

6744

lt125

4380

gt1000

7710

6466

AV5

4961

1000

6675

lt125

5119

971

7710

1239

AV6

4807

1000

6915

lt125

4449

gt1000

6924

0710

AV7

5457

161

6769

250

4555

1000

7042

1250

AV8

4878

1000

6504

125

4217

gt1000

7671

0700

AV9

5226

250

6274

125

4724

1000

7482

8670

AV10

5335

1000

7060

250

4154

100

7404

7720

Cefixime

8693

058

1002

101

5119

058

8693

0200







50(120583gmL)



50(120583gmL)







50value [57] is in



30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)


0

DMSO

Surfa

ctin





(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of





























1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

DPP

H ac

tivity

(a)

1 2 3 4 5 6 7 8 90 1110

A era fractions

0

50

100

150

200

250

300

Ferr

ic io

n re

duci

ng p

ower

(g

AA

eqm

g)

(b)


1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

120

140

160

180

200

Tota

l ant

ioxi

dant

activ

ity (

g A

A eq

mg)




140

120

100

80

60

40

20

0

Mor

talit

y (

)

1 2 3 4 5 6 7 8 9 10

DMSO

Amphoterici

n B







Table3Antibacteria

lactivity

ofA

vera

leafrsquosfractio

ns

Fractio

nsMinim

uminhibitory

concentration(M

IC)

Mluteus

Saureus

Ecoli

Paeruginosa

inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)AV

15793

325

6915

250

5595

673

7600

6020

AV2

4961

1000

6026

500

5702

874

7026

1860

AV3

6314

500

7376

250

4843

1000

8025

1002

AV4

6340

103

6744

lt125

4380

gt1000

7710

6466

AV5

4961

1000

6675

lt125

5119

971

7710

1239

AV6

4807

1000

6915

lt125

4449

gt1000

6924

0710

AV7

5457

161

6769

250

4555

1000

7042

1250

AV8

4878

1000

6504

125

4217

gt1000

7671

0700

AV9

5226

250

6274

125

4724

1000

7482

8670

AV10

5335

1000

7060

250

4154

100

7404

7720

Cefixime

8693

058

1002

101

5119

058

8693

0200







50(120583gmL)



50(120583gmL)







50value [57] is in



30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)


0

DMSO

Surfa

ctin





(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of













1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

DPP

H ac

tivity

(a)

1 2 3 4 5 6 7 8 90 1110

A era fractions

0

50

100

150

200

250

300

Ferr

ic io

n re

duci

ng p

ower

(g

AA

eqm

g)

(b)


1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

120

140

160

180

200

Tota

l ant

ioxi

dant

activ

ity (

g A

A eq

mg)




140

120

100

80

60

40

20

0

Mor

talit

y (

)

1 2 3 4 5 6 7 8 9 10

DMSO

Amphoterici

n B







Table3Antibacteria

lactivity

ofA

vera

leafrsquosfractio

ns

Fractio

nsMinim

uminhibitory

concentration(M

IC)

Mluteus

Saureus

Ecoli

Paeruginosa

inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)AV

15793

325

6915

250

5595

673

7600

6020

AV2

4961

1000

6026

500

5702

874

7026

1860

AV3

6314

500

7376

250

4843

1000

8025

1002

AV4

6340

103

6744

lt125

4380

gt1000

7710

6466

AV5

4961

1000

6675

lt125

5119

971

7710

1239

AV6

4807

1000

6915

lt125

4449

gt1000

6924

0710

AV7

5457

161

6769

250

4555

1000

7042

1250

AV8

4878

1000

6504

125

4217

gt1000

7671

0700

AV9

5226

250

6274

125

4724

1000

7482

8670

AV10

5335

1000

7060

250

4154

100

7404

7720

Cefixime

8693

058

1002

101

5119

058

8693

0200







50(120583gmL)



50(120583gmL)







50value [57] is in



30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)


0

DMSO

Surfa

ctin





(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

DPP

H ac

tivity

(a)

1 2 3 4 5 6 7 8 90 1110

A era fractions

0

50

100

150

200

250

300

Ferr

ic io

n re

duci

ng p

ower

(g

AA

eqm

g)

(b)


1 2 3 4 5 6 7 8 90 1110

A era fractions

0

20

40

60

80

100

120

140

160

180

200

Tota

l ant

ioxi

dant

activ

ity (

g A

A eq

mg)




140

120

100

80

60

40

20

0

Mor

talit

y (

)

1 2 3 4 5 6 7 8 9 10

DMSO

Amphoterici

n B







Table3Antibacteria

lactivity

ofA

vera

leafrsquosfractio

ns

Fractio

nsMinim

uminhibitory

concentration(M

IC)

Mluteus

Saureus

Ecoli

Paeruginosa

inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)AV

15793

325

6915

250

5595

673

7600

6020

AV2

4961

1000

6026

500

5702

874

7026

1860

AV3

6314

500

7376

250

4843

1000

8025

1002

AV4

6340

103

6744

lt125

4380

gt1000

7710

6466

AV5

4961

1000

6675

lt125

5119

971

7710

1239

AV6

4807

1000

6915

lt125

4449

gt1000

6924

0710

AV7

5457

161

6769

250

4555

1000

7042

1250

AV8

4878

1000

6504

125

4217

gt1000

7671

0700

AV9

5226

250

6274

125

4724

1000

7482

8670

AV10

5335

1000

7060

250

4154

100

7404

7720

Cefixime

8693

058

1002

101

5119

058

8693

0200







50(120583gmL)



50(120583gmL)







50value [57] is in



30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)


0

DMSO

Surfa

ctin





(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



Table3Antibacteria

lactivity

ofA

vera

leafrsquosfractio

ns

Fractio

nsMinim

uminhibitory

concentration(M

IC)

Mluteus

Saureus

Ecoli

Paeruginosa

inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)inhibitio

nMIC

(120583gm

L)AV

15793

325

6915

250

5595

673

7600

6020

AV2

4961

1000

6026

500

5702

874

7026

1860

AV3

6314

500

7376

250

4843

1000

8025

1002

AV4

6340

103

6744

lt125

4380

gt1000

7710

6466

AV5

4961

1000

6675

lt125

5119

971

7710

1239

AV6

4807

1000

6915

lt125

4449

gt1000

6924

0710

AV7

5457

161

6769

250

4555

1000

7042

1250

AV8

4878

1000

6504

125

4217

gt1000

7671

0700

AV9

5226

250

6274

125

4724

1000

7482

8670

AV10

5335

1000

7060

250

4154

100

7404

7720

Cefixime

8693

058

1002

101

5119

058

8693

0200







50(120583gmL)



50(120583gmL)







50value [57] is in



30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)


0

DMSO

Surfa

ctin





(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








50(120583gmL)



50(120583gmL)







50value [57] is in



30

25

20

15

10

5

0

Zone

of i

nhib

ition

(mm

)


0

DMSO

Surfa

ctin





(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



(a) (b)

(c)











(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of





(a)

(b)




5 Conclusion







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of







(a)

(b)




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of




Data Availability






Acknowledgments


References







































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of

















































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of
















Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of


Documents

Antioxidant, Antimicrobial, Cytotoxic, and Protein Kinase Inhibition ...downloads.hindawi.com/journals/bmri/2019/6478187.pdf · ResearchArticle Antioxidant, Antimicrobial, Cytotoxic,