1 INTRODUCTION
According to the Inquirer Washington Bureau (2012), they have predicted that major health prolems !ill come "rom the !idespread occurrence o" chronic in"ectious diseases that !ill greatly a""ect the elderly and those !ith lo! immunity# $hese in"ectious diseases lead to increased death rate all over the !orld ased "rom the latest report (%&A $oday 'e!s, 2011)# iseases iseases lie !hooping !hooping cough, uonic plague and tuerculosi tuerculosiss caused y acteria acteria are some speci" speci"ic ic reason reasonss o" this this death death rate rate (*ann, (*ann,200+ 200+)# )# Broad Broad spectr spectrum um antiio antiiotic tic are used used as an e""ective drug against a !ide range o" in"ectious microorganisms !hich includes oth ram positive and ram negative ne gative acteria (&-& .harmaceuticals .ha rmaceuticals ./$# $, $, 2010)# But despite o" this augmentation, augmentation, the World World ealth ealth rgani3ati rgani3ation on reported reported that acteria are ecoming ecoming resistant resistant to these antiiotics (eyes, 2014)# $his is ecause antiiotics has een over5prescried and are used "or a long period o" time ('atural 'e!s, 201 4)# n the other hand, terrestrial terrestrial plant community is est descried descried y its composition composition and dynamics# .lants elonging to terrestrial community indicates their potential role in supplying di""er di""erent ent nutrit nutrition ional al values values and medici medicinal nal uses uses (&huri (&hurin n et al , 2006) due to the presence o" secondary metaolites !ith road iological activities (7lleuch et al., al., 200+)# Anacardium occidentale, occidentale, commonly no!n as 8ashe! contain anacardic acid "ound not only in the cashe! pericarp oil ut also in the nut and the "ruit juice possessing antio9idant activities (:uo et (:uo et al., al., 2006)# $he liquid gained usually distinguish as 8ashe! 'uts &hell iquid (8'&) (8'&) has a road application application use"ul in oth medical purposes purposes and development development o" drugs, drugs, antio9idants, "ungicide, and other chemicals (Andrade et al, 2011)# $he e9tracts !ere evaluated to e9hiit strong antimicroial properties against aga inst gram5positive acteria (imejima et al , 1++1)#
2 In this study, the road5spectrum antiacterial properties o" Anacardium occidentale leaves and pericarp methanolic crude e9tracts !ere evaluated against human pathogens ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Bacillus cereus)# $his leads to the isolation, identi"ication and elucidation o" potent allelochemicals "or drug development#
MATERIALS AND METHODS
Collection of Cashew Leaes and Pericarp Anacardium occidentale leaves and pericarp !ere collected "rom 8ashe! plantation area in uimaras last *ay 2014# 8ollected samples !ere !ashed thoroughly and air dried at room temperature "or t!o days# E!traction of Anacardium occidentale Anacardium occidentale leaves and pericarp !ere then separately soaed in methanol "or t!o days# &les !ere rought to the %niversity o" the .hilippines /isayas, *iag;ao 8ampus, Analytical &ervice aoratory "or in acuo e9traction at <0 08, +0 rpm "or 4 hours# $he solid paste samples !ere !eighed and utili3ed to prepare the concentrations# Preparation of "reatments $he 555g o" solid paste sample "rom the leaves and the pericarp !ere diluted in dimethyl sul"o9ide (*&) to avail the "ollo!ing treatments= $reatment 15 >00 mg?m Anacardium occidentale leaves and $reatment 2 ; >00 mg?m Anacardium occidentale leaves compared to a positive control (tetracycline) and negative control (*&)#
3 Preparation of Culture #edium $he microiological laoratory !as prepared y the in charge and all needed materials !ere provided y the institution# Appro9imately 4@ grams o" *ueller5inton Agar !as diluted in 400m o" distilled !ater in 7rlenmeyer "las and autoclaved "or 16 minutes at 12108 to avail the ideal amount o" medium dispensed in 16 petri dishes# $he melted agar !as allo!ed to cool at <008 e"ore dispensing unto the plates# Preparation of the Bacterial $nocula 8ulture slant o" microes (7scherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Bacillus cereus) !ere provided y the %niversity o" the .hilippines in the /isayas, *iag5ao, Iloilo 8ulture 8ollection# ollo!ing a concentration o" 14 g? o" nutrient roth !as prepared and dispensed in 6 test tues# Aseptically, a loop"ul o" acteria "rom the culture slant !as diluted in the designated nutrient roth and slo!ly s!irled to mi9# $he *carland standard per culture !as achieved y comparing the turidity o" the inocula !ith Barium sul"ate against lac line acground# I" the turidity is cloudy than the standard, nutrient roth !ere added# $he acterial inocula !e re separately and uni"ormly spread using sterile cotton s!a on a sterile .etri dish *ueller5inton agar in a 3ig3ag manner three times eside a lighted alcohol lamp inside the laminar "lo! hood to maintain sterility#
#icrobiological Assay
DMSO (-)
Tetracycline %igure &. esignation o" treatments in the plates#
Cashew leaves extract
Cashew pericarp
4 A"ter all the plates !ere s!aed !ith the acteria, the discs !ere put on the agar on the designated points# $he agar plugs !ere removed and sterili3ed in ysol solution# In vitro antiacterial activity test !as carried out y dis di""usion method# $o test the antiacterial activity, Anacardium occidentale leaves and nut shell crude e9tracts using various solvent !ere dispensed in disc at 10 in a cloc!ise manner# A"ter all the treatments !ere dispensed the controls, the .etri plates !ere placed inside the incuator "or 1@ hours at 4608 temperature# -esults !ere ased on the diameter o" the 3one o" inhiitions o" test treatments and the controls# Antimicroial Inde9 !as determined to interpret the e9tractCs activities# By comparing the areas o" 3one o" inhiition o" test !ith standard the concentration and potency o" test samples are determined (u.ages, 2014)# Inhiition o" the acterial gro!th !as measured in millimeters# 'ata and 'ata (athering Procedures A"ter an overnight incuation, the .etri plates !ere inspected "or structure o" 3ones o" inhiition around the "ilter paper discs# 'iameter of one of inhibition $he diameter o" the 3ones !ere measured y means o" a ruler and the average data !as taen "rom the data in the 9 and y a9is o" the 3one# $he data !as plotted in taulated "orm and treated statistically to determine the antimicroial Allelochemicals present in Anacardium occidentale leaves and nut shell against "ive acteria namely, E. coli, Staphyloccocus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus cereus. Statistical 'ata Analysis Procedure $he data otained "rom the study !ere sujected to the "ollo!ing descriptive and in"erential statistical treatments using the &tatistical .acage "or &ocial &ciences (&.&&)
5 &o"t!are# $he statistical tools used in this study !ere= *ean5 $he mean !as used to e9press the average diameter o" the 3one o" inhiition developed in each treatment in this study# &tandard deviation5 $o determine the dispersion et!een the mean# A'/A5 used to determine the signi"icant di""erence o" three or more groups means set at 0#06 level o" signi"icance# uncanCs *ultiple -ange $est (*-$) ; compare signi"icant di""erences o" means o" various groups or treatments in the study# Plant identification 8ollected plant samples o" cashe! leaves and pericarp !ere rought to the %niversity o" the .hilippines, *iag5ao, Iloilo "or plant identi"ication#
RESULTS
$he diameter o" the clear 3one o" inhiition around the impregnated dis dispensed on the sur"ace o" the agar indicates antiacterial activities o" the e9tracts eing tested# $hese results are compared to the t!o standardsD one is the positive control tetracycline a no!n antiiotic and a negative control, dimethyl sul"o9ide= the diluent used to prepare the various concentrations# $he quantitative data is e9pressed in millimeter and "urther analy3e o" itCs the potency y determining the Antimicroial Inde9 (AI)# $he closer the value o" AI to 1 the more potent is the sample# $his
is the asis "or "urther io5directed antiacterial assay "or the detection o" secondar y metaolites as antiiotic# $he antiacterial activities o" the samples against E. coli sho!ed that the cashe! leaves and the pericarp methanol crude e9tracts e9hiited resistant activities comparale !ith the *&# $etracycline eing a no!n antiacterial agent e9hiited 1<##60 "ollo!ed y $etracycline having a mean o" 20#
! $he antiacterial activities o" the samples against Bacillus cereus sho!ed that the 8ashe! pericarp methanol crude e9tract revealed the highest antiacterial activity having a mean o" >4#44 mm "ollo!ed y $etracycline having a mean o" 26#00 mm !hile negative activity e9hiited y the 8ashe! leaves and *&# ne5!ay analysis o" variance and uncanCs test revealed signi"icant di""erence in the mean 3one o" inhiitions against Bacillus cereus having a p5 value o" #000F0#06 level o" signi"icance# $he antiacterial activities o" the samples against Bacillus subtilis sho!ed that the 8ashe! pericarp methanol crude e9tract revealed the highest antiacterial activities having a mean o" ><#00 mm "ollo!ed y $etracycline having a mean o" 4+#00 mm !hile negative activity e9hiited y the 8ashe! leaves and *&# ne5!ay analysis o" variance and uncanCs test revealed signi"icant di""erence in the mean 3one o" inhiitions e9hiited y the cashe! pericarp against Bacillus subtilis having a p5 value o" #000F0#06 level o" signi"icance#
Samples
Samples Clear Zone Diameter (mm) SD 1
Cashew Leaves Escherichia coli
Cashew pericarp
DMS (!) "etrac#line ($)
2
3
0 0 0 1%
0 0 0 1%
0 0 0 1&
0 3 0 20
0 3+ 0 1%
0 30 0 2
Mean* 0b 0b 0b 16.67a
AI**
0 0 0
0 0 0 1.78
0
0 4.50 0 2.45
p val'e 000
Cashew Leaves Pseudomonas aeruginosa
Cashew pericarp
DMS (!) "etrac#line ($)
0c 33.00a 0c 20.67b
0
" p value = .000
Cashew Leaves Staphylococcus aureus
Cashew pericarp
DMS (!) "etrac#line ($)
0 30 0 31
0 2 0 1
0 3 0 13
0 0 0 20
0 0 0 3
0 30 0 20
0 + 0 3
0 3 0 3%
0 , 0 +
0c 30.00a 0c 19.67b
0
0c 43.33a 0c 25.00b
0
0c 46.00a 0c 39.00b
0
0
0 4.00 0 2.28
p val'e 000
Cashew Leaves Cashew pericarp
Bacillus cereus
DMS (!) "etrac#line ($)
0
0 6.22 0 3.17
p val'e 000
Cashew Leaves Bacillus subtilis $ale 1
Cashew pericarp
DMS (!) "etrac#line ($)
0
0 6.67 0 5.50
p val'e 000
Antibacterial actiities of Cashew leaes and shell e!tracts on selected human pathogens compared to the control and its antimicrobial inde! *A$)
* Col'mn means with common letter s'perscripts per -acteri'm are not si.ni/icantl# i//erent at 00 level o/ si.ni/icance -# ne!wa# AA an D'ncans test **Antimicro-ial Ine4 1 or 5 than
is potent antimicro-ial a.ent
#
%igure +# -evealed clear 3ones o" inhiition around the impregnated dis o" the samples used= (A) E. coli, (B) P. aeruginosa, (8) S. aureus, () B. cereus and (7) B. subtilis.
DISCUSSION
$he 3ones o" inhiitions !ere oserved around the discs, this indicated antiacterial activities o" the plant leaves and pericarp e9 tracts# $he sensitivity o" the di""erent test organisms to methanolic e9tracts o" the leaves and pericarp o" Anacardium occidentale !as sho!n y 3one o" inhiitions a"ter 1@ hours o" incuation(As illustrated in igure 1)# $he asence o" 3one o" inhiitions around each discs signi"ied resistance# It !as oserved that the crude e9tracts o" Anacardium occidentale leaes did not inhiit the gro!th o" any o" the test acteria# While the Anacardium occidentale pericarp e9tract e9hiited potent or strong antiacterial properties against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis higher than the positive control ; tetracycline# $his indicates that the 8ashe! pericarpl is rich in allelochemicals or secondar y metaolites that e9hiited the antiacterial properties#
1$ According to 'andi (2014), the cashe! shell contains reddish ro!n oil and high 3inc concentration# $his concurs !ith the !or o" :annan et al. (200+) sho!ing that it has various range o" iological properties and distinct range o" p hytochemicals present# 8ontrastingly to the study o" Agedah et al. (Bioline, 2010), the leaves e9tract e9hiited antiacterial activities against E. coli and S. aureus. $he antiacterial assay o" 8ashe! pericarp and leaves provide strong indication that the pericarp contains allelochemicals !ith road5spectrum antiacterial activities# $his result also encourages the isolation, characteri3ation and elucidation o" allelochemicals !ith potent antiacterial activities# 8onsidering that today a lot o" diseases are attriuted to the prevalence o" in"ections caused y these range o" human pathogens# $he "ormulation and production o" end products "or !ide array o" medicinal, medical and industrial uses "rom the 8ashe! pericarp is also e9pected "rom this providing aseline in"ormation "or in5depth research#
CONCLUSIONS
$he cashe! pericarp methanolic crude e9tract contain allelochemicals !ith potent antiacterial properties against ram positive and ram negative human pathogensD S. aureus, P. aeruginosa, B. cereus and B. subtilis e9cept E. coli. n the other hand, the cashe! leaves methanolic crude e9tract e9hiited negative an tiacterial properties against all tested human pathogens# -esults lead to "ormulation o" several products !ith !ide array o" medicinal uses and identi"ication o" the potent allelochemicals#
11
References Cited
Andrade, $# , AraGjo, B#, 8itH, A# *# , &ilva, #, -ichter *# # , erra3, A# (2011) Antio!idant properties and chemical composition of technical Cashew ut Shell Li-uid *tCSL)# -etrieved last &eptemer E, 2014 "rom http=??academic#research#microso"t#com?.ulication?>+44+@2>?antio9idant5 properties5and5chemical5composition5o"5technical5cashe!5nut5shell5liquid5tcnsl 7lleuch, # , &haaan, *# , &maoui, &# , *ellouli, # , -eai, I# :#, Ben guira, # # , &haaan, :# A# , and aatsch, # ( 200+) Bioactie Secondary #etabolites from a ew "errestrial Streptomyces sp. "++. -etrieved last &eptemer E, 2014 "rom http=??!!!#nci#nlm#nih#gov?pmc?articles?.*8412@E0>?
%eyes & ' D& (2$13) The end of Modern Medicine has arrived, according to the WHO etrieve* pril 11& 2$13 ,r. http/00wwwnatralnewsc.0$35"#$antiiticsresistance%Oht.l
12 imejima *# and :uo I# (1++1) Antibacterial Agents from the Cashew Anacardium occidentale *Anacardiaceae) ut Shell /il. -etrieved last &eptemer E, 2014 "rom http=??!!!#aseaniotechnology#in"o?astract?2100002+#pd" u.ages, (2014) #icrobial 0 #icrobiological assay1 $ts 'efinition 2ses and methods# -etrieved last April 11, 2014 http=??heem#hupages#com?hu?*icroial5*icroiological5assay5Its5e"inition5%ses5 and5methods :uo, *asuoa, a, $sujimoto , (2006) Antio!idant actiity of Anacardic acids# -etrieved last April 11, 2014 http=??!!!#agronavigator#c3?user"iles?ile?Agronavigator?:vasnicova?anacardic J20acids#pd"
Mann& D& (2$$#) Study: Showerheads may deliver blast of bacteria. etrieve* last Septe.er !& 2$13 ,r.
http/00e*itincnnc.02$$#0%6T%0$#0140shwerhea*acteria0in*ex ht.l
7S T*ay& (2$11 ) WHO's to !illers: Heart roblems, infectious diseases, cancer etrieve* last pril 11& 2$13 http/00sat*ay3$sat*ayc.0news0health02$$"-1$-2!-wh-*eathreprt8ht. Shrin& ' 9& :rner : S& an* %illeran* % (2$$5 " #ll wet or dried u$ %eal di&erences between auatic and terrestrial food webs. etrieve* last Septe.er !& 2$13 ,r. http/00wwwncinl.nih;v0p.c0articles0
ACKNOWLEDGEMENT
%tmost gratitude is e9tended to the "ollo!ing persons=
13 *rs# loria 8armencita &# imoto, *r# ione # uman and *rs# -osene *# uman, parents o" the researchers# *r# eandro amarcha and *rs# *a# $heresa -omey o" the 8ollege o" Arts and &ciences, %./5 *iag5ao 8ampus# *rs#-ose 7angcol o" &iunag, uimaras "or providing the samples# *r# -onilo /# Aponte and *rs# Branche A# &umido, -esearch Advisers *s# race *amon and *r# 'eil ohn oquiKo, the researchersC grammarian and statistician# *rs# $oni inah 8heer ernande3, epartment ead o" &chool o" the uture (&), Iloilo 'ational igh &chool# *r# 7d!in # *osura, -esearch ead5 -esearch epartment, Iloilo 'ational igh &chool# r# 'ordy # &iason r# .rincipal I/5 Iloilo 'ational igh &chool *ost o" all, to the Almighty od "or the strength and !isdom provided to the researchers#
