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TABTABA (Nostoc linckia) ETHANOLIC EXTRACTS AGAINST

STAPHYLOCOCCUS AUREUS
Nero M. Paderes
Researcher
ABSTRACT
This study was conducted at ASIST, Main Campus, Lagangilang, Abra from August !"#
to $une !"%& It aimed to determine the anti'Staphylococcus aureus acti(ity of ethanolic e)tracts
of tabtaba *nostoc linckia+& Specifically, it aimed to determine the anti'staphylococcal acti(ity of
tabtaba *nostoc linckia+ ethanolic e)tracts at ,-, ,!-, and .,- concentration in terms of the
diameters of growth inhibition, the significant difference between and among the diameters of
growth inhibition of the abo(e'mentioned organism sub/ected to the different e)tract
concentrations, and to determine which of the three concentrations has the highest potential of
ha(ing anti'Staphylococcus aureus properties&
The study was laid out using the Complete Randomi0ed 1esign *CR1+& The grand mean
was computed among the different treatments and the data were analy0ed using A234A&
1uncan5s multiple range tests *1MRT+ was used to determine the significant differences between
the compared means&
Result shows that at ,-, ,!-, and .,- concentration, tabtaba ethanolic e)tract
e)hibited a strong antibacterial acti(ity against S& aureus with a mean 0one of inhibition of ".&.6
mm, "&.6 mm, and "6&!% mm respecti(ely, while the positi(e control *7enicillin+ showed a
strong antibacterial acti(ity against S& aureus with a mean 0one of inhibition of #"&6. mm& This
shows that tabtaba ethanolic e)tracts has a strong anti'staphylococcus aureus properties&
Among the three treatments, fifty percent *,!-+ concentration of tabtaba e)tract
produced the highest mean 0one of inhibition with a difference of #&8 mm with that of ,-
concentration and #&., mm with that of .,- concentration&
A234A shows a highly significant differences among the different treatments of
ethanolic e)tracts against S& aureus *"!&!",+& This indicate that the organism is highly sensiti(e
to the inhibitory effect of the different ethanolic e)tracts& Based on the 1MRT result, it shows
that there is no significant difference on the effect of ,- and .,- concentrations of tabtaba
e)tracts but at ,!- concentration there is significant effect of Tabtaba e)tracts towards
Staphylococcus aureus&
The researcher recommends another study to be conducted against other microorganisms
in order to obtain more (alid information on the anti'microbial acti(ity of tabtaba&
I& Rationale
The continuous reappearances of Staphylococcus aureus infection continue locally within
the pro(ince of Abra particularly in the upland municipalities where health and sanitation is still
below the acceptable norms& Staphylococcus aureus is found on the s9in and in the nostrils of
many healthy indi(iduals& These bacteria often gi(e rise to minor superficial diseases, including
the formation of pustules or boils in hair follicles& Staphylococcus aureus infections are
characteri0ed by the presence of pus and formation of abscesses& In addition to s9in pustules,
boils, and carbuncles, Staphylococcus aureus is responsible for impetigo, infections of wounds
and burns *particularly in a hospital en(ironment+, breast abscesses, whitlow *inflammation of a
finger or toe near the nail+, osteomyelitis, bronchopneumonia, septicemia, bacteremia, acute
endocarditis, food poisoning, and scalded s9in syndrome& Scalded s9in syndrome occurs in
newborns and is due to infection by to)igenic strains of S. aureus. The to)ins cause the s9in to
e)foliate, which lea(es an appearance of ha(ing been scalded *Microsoft :ncarta, !!6+&
;ith antibiotic resistance increasing dramatically, the treatment of S& aureus infections
becomes more difficult imposing public health threats, and economic burdens on health care
costs *Snowden et al, !"%+& The antibiotic drug penicillin was once effecti(e for the treatment
and control of staphylococci, but the increase of resistant strains has re<uired use of other
antibiotic agents such as semi'synthetic penicillins, cephalosporins or (ancomycin& Studies
announced in !!= showed that the antibiotic daptomycin was effecti(e against bloodstream and
heart infections as well as against s9in infections *Microsoft :ncarta, !!6+&
3ne way of controlling antibiotic resistance is the introduction of alternati(e forms of
anti'staphylococcus aureus medication which are commonly found in nature& And one of these
nature gifts is in the form of a blue'green algae commonly 9nown as Tabtaba or also 9nown as
bakbakasi, barbaradio, badbadiok, dodoldol, kulatt, and bakatel in other places&
The scientific name of the locally growing species in 2orthern Lu0on is Nostoc linckia& A
nostoc colony is ordinarily not seen> but after a rain it swells up into a conspicuous /ellyli9e
mass& Tabtaba is usually har(ested in the fields /ust after the rice were cut and?or threshed when
the rice field is somewhat dry, these bounty of the earth lay in the ground abundantly, waiting to
be pic9ed*http@??pina9betrepublic&blogspot&com+&
Currently, there is none or few published data on the Anti'Staphylococcus aureus acti(ity
of ethanolic e)tracts from Tabtaba or Aresh ;ater Blue' Breen Algae *Nostoc linckia+&
Therefore, the researcher determined if this species of fresh water algae can be a potential source
of anti'staphyloccus aureus medication& Cence, this study was concei(ed&
II& Re(iew of Related Literature
Resurgences of Staphylococcus aureus infection continue globally, with antibiotic
resistance increasing dramatically, ma9ing these infections more difficult to treat&
Staphylococcus aureus epidemics impose public health threats, and economic burdens on health
care costs worldwide, presenting challenges which modern medicine struggles to control
*Snowden, !"%+& 3ne way of reducing resistance is with the use of naturally occurring
antibiotics that is usually deri(ed from plant e)tracts as well as from other sources such as algae&
Algae ha(e recently recei(ed significant attention in the search for bioacti(e compounds
to de(elop new drugs and health foods *$in Diao, !"!+& A detailed screening of micro'algae in
marine as well as freshwater systems ha(e re(ealed a whole new range of molecules with
antibiotic, anti(iral and anticancer acti(ities as well as anti'inflammatory, hypocholesterolaemic,
pharmacological properties *Borowit09a, "888+& This growing interest in algae and algal products
has led to e)traction of many important compounds with promising biological acti(ity from
potentially useful organisms *Beena B& et al&, !""+&
4arious researches re(ealed that antibacterial acti(ity of freshwater algae has been
obser(ed on different bacterial strains& A study conducted by Beena B& et al *!""+, re(eals that
Scenedesmus sp& can be used as a potential source of antibacterial compounds against pathogenic
strains if e)tracted effecti(ely with suitable sol(ents&
3ther study shows that methanolic e)tract of 3& sancta a fresh water algae showed
antibacterial acti(ity against three pathogens *7& mirabilis, 7& (ulgaris and S& aureus+ with
inhibition 0ones of "!, 6, and 6 mm , while methanolic e)tracts of S& decimina e)hibited
antibacterial acti(ity against S& aureus and 7& mirabilis with a 0one of inhibition of " and 8 mm
respecti(ely *$ustella ;&7&,et al&,!""+&
A study on freshwater algae two Anabaena species were tested for its antimicrobial
acti(ity with the agar well diffusion method against gram positi(e bacteria Staphylococcus
aureus& In (itro study of antibacterial acti(ity of different organic e)tracts of Anabaena sp& Is
done& The acti(ity was assessed by using a<ueous, acetone, methanol and he)ane e)tracts& These
e)tracts showed specific acti(ity in inhibiting the growth of Staphylococcus aureus& The
methanolic e)tract showed largest 0one of inhibition compared to other e)tracts& Their results
indicate that these species has a significant capacity for being used as potent natural antibiotics
*Shantanu, !"#+& In the .!s, the 2IST pursued further research on culti(ation of the nitrogen'
fi)ing blue'green algae with emphasis on edible forms commonly utili0ed by nati(es in 2orthern
Lu0on& 3ne filamentous edible species, 2ostoc linc9ia, locally called tabtaba was studied
*Rodulfo "86!+& This alga grew best in a nitrogen'free inorganic medium, had a protein content
of %!' %,- *dry weight+, and grew well in an al9aline pC *.&,'6&!+ *Rodulfo, B&R&,"88!+&
III& 3b/ecti(es of the Study
This study aimed to determine the anti'Staphylococcus aureus acti(ity of ethanolic
e)tracts of tabtaba *nostoc linckia+&
Specifically, this research study aimed to@
"& 1etermine the anti'staphylococcal acti(ity of tabtaba *nostoc linckia+ ethanolic e)tracts
at ,-, ,!-, and .,- concentration in terms of the diameters of growth inhibition&
& 1etermine if there is a significant difference between and among the diameters of growth
inhibition of the abo(e'mentioned organism sub/ected to tabtaba *nostoc linckia+
ethanolic e)tracts at ,-, ,!-, and .,- concentration, and
#& To determine which of the three concentrations of tabtaba *nostoc linckia+ ethanolic
e)tracts has the highest potential of ha(ing anti'Staphylococcus aureus properties&
I4& Methodology
A& Materials
The materials used in the study were as follows@
Tabtaba (Nostoc linckia), Staphylococcus aureus, Tap water and sterili0ed
distilled water, :thanol, Mortar and pestle, digital weighing scale, o(en, airtight
container, sterili0ed glass bottles, bea9er, :rlenmeyer flas9, filter paper,
e(aporating dish, magnetic stirrer hotplate, fume hood, 7enicillin dis9s
*"mg?ml+, Mueller Cinton Agar, o(en, ;hatmann5s 2o&" filter paper for the disc,
portable autocla(e, forceps, inoculating loop, inoculating hood, digital weighing
scale, laboratory gowns, glo(es, mas9, and cap, graduated cylinder, bea9er,
:rlenmeyer flas9, and test tube with caps, and alcohol lamp&
B& Methods
"& Collection of Tabtaba
Tabtaba *Nostoc linckia+ was collected from Lagangilang and Bucay Abra&
& 7reparation of Tabtaba (Nostoc linckia) e)tracts
The tabtaba algae was washed thoroughly under running tap water to remo(e dirt
and then rinsed with distilled water& It was o(en dried at =!EC and powdered using
mortar and pestle and stored in airtight containers until further used& !! grams of
powdered test plants was placed in separate sterile glass bottles and ,!! ml of ethanol
was added to completely soa9 the plant samples& Samples was soa9ed for , days
*2wachu9wu et&al&!""+&
#& Ailtration
After , days of soa9ing, the separated e)tracts *,!! ml+ was filtered using
;hatman5s no& " filter paper and placed in an :rlenmeyer flas9& *2wachu9wu
et&al&!""+&
%& 1rying
The ethanol filtrate was condensed to dryness using a magnetic stirrer hotplate&
The separated plant e)tracts was indi(idually concentrated into ,! ml and stored in a
(ial at %EC for further e)perimental procedures& *2wachu9wu et&al&!""+&
,& 7reparation of different concentration of e)tracts
The plant e)tracts was measured using a pipette and a bea9er& Aor T", the positi(e
control *7enicillin dis9+ that was used will be at " mg?ml> T will be measured
depending on the amount of concentration& In preparing a solution of .,-
concentration, ., ml of the e)tract will be added to "!! ml of sterile distilled water>
while in preparing a solution of ,!- concentration, ,! ml of e)tract will be added to
"!! ml of sterile distilled water> and in preparing a solution of ,- concentration, ,
ml of e)tract will be added to "!! ml of sterile water *Celmenstine A&+&
=& Laboratory materials and filter paper disc preparation
Ailter paper disc was prepared using a paper puncher to produce e<ual disc si0es&
Laboratory materials was sterili0ed using a portable autocla(e while filter paper dis9s
was sterili0ed using an o(en sterili0er for ! minutes at "6!EC&
.& 7reparation of culture media and inoculum
7ure culture of test organisms was sub'cultured onto Muller Cinton culture media
using the Firby'Bauer method& The culture media was prepared by adding &% grams
of agar in e(ery "!! ml of sterili0ed distilled water, autocla(ed and allowed to cool up
to %,EC& Se(enty fi(e *.,+ ml of Muller Cinton agar was separately poured into plates
and uniformly inoculated with Staphylococcus aureus using a sterile inoculating loop
by means of the strea9 plate method& This was performed inside an inoculating
chamber to maintain sterility and to a(oid contamination of the culture plates&
7repared bacterial cultures was incubated for % hours at #,GC& Bacterial colonies was
obser(ed after the incubation period& These were prepared at the Biology Laboratory
of Abra State Institute of Science and Technology, Main Campus&
6& Antibacterial Assay
The test was determined by using the Firby'Bauer disc diffusion *Bauer et& al&
"88=+& This method was performed by means of soa9ing filter paper discs ha(ing the
same si0es into the different concentrations *.,-, ,!- and ,-+ of Tabtaba *2ostoc
linc9ia+ ethanolic e)tracts& Se(enty fi(e *.,+ ml of culture media agar was poured into
separate plates and was uniformly inoculated with Staphylococcus aureus organisms
using a sterile inoculating loop by means of the strea9 plate method& By using a
flame'sterili0ed forceps, the soa9ed filter discs of algae e)tracts together with the
7enicillin dis9 *positi(e control+ was placed on the top of the seeded culture agar& The
plates were incubated o(ernight at #,EC& The growth of inhibition was measured in
millimeter using a digital 4ernier caliper and the effects of the different treatment was
compared with that of the positi(e control& The anti'staphylococcus aureus acti(ity
testing were done in three replications&
C& Research 1esign
Complete Randomi0ed 1esign *CR1+ was be used in this study& The following
treatments were@
T!' positi(e control *7enicillin "mg?ml+
T"' , - Tabtaba ethanolic e)tract concentration
T' ,! - Tabtaba ethanolic e)tract concentration
T#' ., - Tabtaba ethanolic e)tract concentration
1& 1ata Bathering Instrument
The researcher obser(ed and recorded the 0one of inhibition that was produced by
the different concentration of Tabtaba e)tracts at .,-, ,!-, and ,- in millimeter *mm+
using a 4ernier Caliper and compared them with the positi(e control& The descripti(e
interpretation for the range of inhibition was adopted from 3ngtengco *"88+&
Table "
Range of Standard Hone of Inhibition in mm
Hone of Inhibition Inhibitory Acti(ity
I ".
" J "=
. J ",
= or !
K K K, strong
K K, moderate
K, wea9
J , negati(e
:&Statistical Analysis of 1ata
To interpret the 0one of inhibition the following statistical tools was utili0ed@
"& A(erage Mean' was used to determine the a(erage growth of inhibition produced
by the positi(e and negati(e control and of the different concentrations *.,-, ,!-,
and ,-+ of Tabtaba *2ostoc linc9ia+ e)tracts&
& Analysis of 4ariance *A234A+' was used to determine if there e)ist a significant
differences in the a(erage mean of the growth of inhibition treated under the positi(e
control and the different concentrations of Tabtaba *2ostoc linc9ia+ e)tracts& A234A
was sub/ected at !&!, le(el of significance&
#& 1MRT' 1uncan5s multiple range tests was used to determine the significant
differences between the compared means&
4& 1iscussion of Results
Tabe !. S"##ar$ o% Mea% &o%es o' I%h(b()(o% o' )he *(''ere%) Co%ce%)ra)(o%s o' Tab)aba
(Nos)oc (%c+(a) a) !,-. ,/- a%d 0,- A1a(%s) S)a2h$ococc"s a"re"s
Test 3rganism Treatments Brand Mean of
Hone of Inhibition
1escripti(e
Interpretation
Staphylococcus aureus
Control *7enicillin+
,- concentration
,!- concentration
.,- concentration
#"&6. mm
".&.6 mm
"&.8 mm
"6&!% mm
KKK, Strong
KKK, Strong
KKK, Strong
KKK, Strong
Hone of inhibition ha(ing I ". mm is considered *KKK+ strong
The table shows that at ,-, ,!-, and .,- concentration, tabtaba ethanolic e)tract
e)hibited a strong antibacterial acti(ity against S& aureus with a mean 0one of inhibition of ".&.6
mm, "&.6 mm, and "6&!% mm respecti(ely, while the positi(e control *7enicillin+ showed a
strong antibacterial acti(ity against S& aureus with a mean 0one of inhibition of #"&6. mm& This
shows that tabtaba ethanolic e)tracts has a strong anti'staphylococcus aureus properties& The
present study agrees with the wor9 of 1hanala9shmi M&,et al, !"#, their study shows that
Chlorophycean members ' 1esmococcus oli(aceous, chlorococcum humicola and Chlorella
(ulgaris in showing the presence of al9aloids, cardiac glycoside, terpenoids and saponins& These
phytoconstituents were also earlier reported for their antimicrobial acti(ity& In this conte)t the
acti(e metabolites present in ethanol e)tract of the algae may be associated with their
antibacterial property&
Among the three treatments, fifty percent *,!-+ concentration of tabtaba e)tract
produced the highest mean 0one of inhibition with a difference of #&8 mm with that of ,-
concentration and #&., mm with that of .,- concentration& The result is parallel to the study of
2wachu9wu, et&al *!""+, that the higher the concentration of the constituents does not always
mean higher acti(ity of 0one of inhibition&
A234A shows a highly significant differences among the different treatments of
ethanolic e)tracts against S& aureus *"!&!",+& This indicate that the organism is highly sensiti(e
to the inhibitory effect of the different ethanolic e)tracts& This can be substantiated by the study
of 2edel9os9a et, al *!"#+, that gram K organisms such as S& aureus are more sensiti(e to plant
e)tracts&
1MRT result shows that there is no significant difference on the effect of ,- and .,-
concentrations of tabtaba e)tracts but at ,!- concentration there is significant effect of Tabtaba
e)tracts towards Staphylococcus aureus&
4I& Conclusion
Based on the result of the study, the following conclusions are drawn@
"& All concentrations of tabtaba ethanolic e)tracts *,-, ,!- and .,-+ ha(e strong anti'
staphylococcus aureus acti(ity&
& The most potent among the # e)tracts is at ,!- concentration which produced the highest
mean 0one of inhibition&
4II& Recommendation
The researcher recommends another study to be conducted against other microorganisms
in order to obtain more (alid information on the anti'microbial acti(ity of tabtaba&
4III& Bibliography
Beena B& et al&, Antibacterial Acti(ity of Areshwater Microalga *Scenedesmus sp&+ Against Three
Bacterial Strains, $& Biosci&, !""& 4ol& *%+@"=!'"=,&
Borowit09a, M& A&, $& Appl& 7hycol& "888, ., #'",&
$ustella ;&7&,et al&,Antimicrobial Acti(ity of Certain Aresh ;ater Microalgae Arom
Thamirabarani Ri(er, Tamil 2adu, South India, Asian 7acific $ournal of Tropical
Biomedicine&, pS".!&
Shantanu,B&, Antimicrobial Acti(ity of Two 1ia0otropic Cyanobactera Against Staphylococcus
aureus&Int& $& Med,Arom 7lants, 4ol& #, 2o& , pp& 6#'8, $une !"#&
Rodulfo, B&R& *"88!+& Culture and utili0ation of freshwater algae as protein source& In@ I&$&
1ogma $r&, B&C& Trono $r&, L R&A& Tabbada *:ds&+ Culture and use of algae in Southeast
Asia@ 7roceedings of the Symposium on Culture and Mtili0ation of Algae in Southeast
Asia, 6'"" 1ecember "86", Tigbauan, Iloilo, 7hilippines& *pp& 6"'6.+& Tigbauan, Iloilo,
7hilippines@ A<uaculture 1ept&, Southeast Asian Aisheries 1e(elopment Center&
IN& Appendi)
Tabe 3. O%e45a$ ANO6A o' )he Mea% &o%es o' I%h(b()(o% o' )he d(''ere%)
co%ce%)ra)(o%s o' Tab)aba e7)rac)s a1a(%s) S)a2h$ococc"s a"re"s.
Source of
4ariance
1egree of
Areedom
Sum of S<uare Mean S<uare Computed A
Treatment # #8"&!!8 "#!&##= "!&!"#OO
:)perimental
:rror
6 6&=66 "&!6=
Total "" #88&=8.
C&4& P %&.-
OO Significant "- le(el
Tabe 8. *"%ca%9s M")(2e Ra%1e Tes) o' )he Mea% &o%es o' I%h(b()(o% o' *(''ere%)
Tab)aba e7)rac)s a1a(%s) S)a2h$ococc"s a"re"s
Treatment Mean
Control #"&6.
a
,- ".&.6
c
,!- "&.8
b
.,- "6&!%
c
OMeans with the same letter are not significant
Tabtaba Collection
Soa9ing in :thanol
Ailtration of :)tracts
7reparation of different concentrations
Bacterial inoculation
Bacterial Assay