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RESEARCH PAPER OPEN ACCESS
Assessmen o an imic obial ac i i y o lea es ex ac o
Suaeda
u icosa
agains clinically impo an bac e ial s ains
Kaina Aami , Abdul Rehman*
Depa men o Mic obiology and Molecula Gene ics, Uni e si y o he Punjab, New Campus,
Laho e 54590, Pakis an
Key wo ds: Disc di usion me hod, Aga well di usion me hod, Suaeda u icosa, Zone o inhibi ion, Minimum
bac e icidal concen a ion.
h p://dx.doi.o g/10.12692/ijb/16.6.129-139
A icle published on June 16, 2020
Abs ac
Plan s ha e played a signi ican ole in he sc eening o bioac i e compounds o human medica ions. The aim o
p esen s udy was o assess he an ibac e ial ac i i y o Suaeda u icosa lea es ac ions agains clinically
impo an bac e ial s ains. The ex ac o S. u icosa lea es was o med in di e en sol en o di e en pola i y
and hei an imic obial ac i i y was es ed agains ou pa hogens i.e. Esche ichia coli, Klebsiella pneumoniae,
Pseudomonas ae uginosa and Salmonella en e ica h ough Disc di usion assay and aga well di usion me hod.
Quali a i e phy ochemical analysis was pe o med o he p esence o bac e icidal ac i e compounds. Resul s
ob ained clea ly demons a ed he e icacy o me hanolic ex ac , dime hyl sul oxide il a e, and la onoid
ac ion agains pa hogenic bac e ial s ains. The phy ochemicals ex ac ed om lea es ac ed asb oad-spec um
ac i e compounds agains hese pa hogens. These ex ac s had highes po en ial agains E. coli wi h lowes
minimum inhibi o y concen a ion o 60 µl/5ml. The lea es o S. u icosa ac as po en o igin o nume ous
phy ochemicals ha can make headway o an imic obial d ugs.
* Co esponding Au ho : Abdul Rehman [email protected]
In e na ional Jou nal o Biosciences | IJB |
ISSN: 2220-6655 (P in ), 2222-5234 (Online)
h p://www.innspub.ne
Vol. 16, No. 6, p. 129-139, 2020
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In oduc ion
Bac e ia ha e abili y o de elop esis ance agains
mul iple an ibio ics wi h he passage o ime and we
a e swi ly p oceedings owa ds an e a when
an ibio ics will no wo k e icien ly on bac e ial
in ec ions (Giou ie a e al., 2019). New an i-mic obial
compounds ha e been equi ed o human ela ed
heal h issues. Resis ance o an ibio ics is one o he
bigges p oblems ha ace common heal h issue
(Mah, 2019).
In o de o ind new an imic obial agen s wi h a no el
mode o ac ion, plan s ha e been disco e ed as a
sou ce o he ecogni ion o new and e ec i e
an imic obials (Sanie al., 2019). He bal medicines
a e he oldes o m o heal h ca e.
The he bal medicine in e ac s wi h di e en
cons i uen s o inc ease hei ac i i y. In de eloping
coun ies, he demand o he bal plan s is inc easing
day by day because hey do no ha e any side e ec s
(Mzoughie al., 2018). I has p o ed o be a g ea
sou ce o bene i s o he poo . Now a day he e has
been a su icien ly g ea demand in he use o
medicinal p oduc as a emedial agen and
phy ochemicals compounds (Pe opoulos e al.,
2018).
In adi ional medicines, he he b Suaeda u icosa,
is also known as a sh ubby sea bligh , is a woody
halophy e abou 60-90cm all. Suaeda is de i ed
om an A abic wo d “Suwaid” which means black
(Llanes e al., 2018). I has been used o gas o
en e i is, s omach pain, wound and skin in ec ion,
ea men o sinusi is, dia hea, in an ile eczema and
ube culosis. Many plan s ha e been used un il now
due o hei bac e ios a ic and bac e icidal na u e.
They con ain seconda y me aboli es ha a e e y
impo an om an indus ial poin o iew. These
ac i e compounds we e named as alkaloid, phenol,
la onoid, e penoid, and s e oid(A ia-Ismail, 2016).
S. u icosa ex ac has been epo ed o
hypoglycemia and hypolipidemia ac i i ies. I can be
used o oo hache and ch onic heuma ic hea
disease. Plan ex ac exhibi s some cha ac e is ics
ha ep esen an an i- i al agen agains hepa i is. I
is used as an oin men o emo e excess a and
educes oil salini y (Öz ü k e al., 2019).
Phy ochemicals a e able o esis pep idoglycan
syn hesis, damage he mic obial memb ane s uc u e,
al e he bac e ial memb ane su ace hyd ophobici y
and also modula e quo um sensing (Janua i e al.,
2019). Phenols a e dis inguished o hei an i- ungal,
an i-cance ous, an i-oxidan and an i-mic obial
ac i i ies. These molecules can easily pene a e due o
hei low pola i y h ough he cell memb ane (Sk oza
e al., 2019). Alkaloids ac as allelochemicals, au o-
induce s, and side opho es. Thei mode o ac ion is o
inhibi he nucleic acid syn hesis, as hey a ge he
enzyme ac i i y o dihyd o ola e educ ase.
They a e espi a o y inhibi o s as hey lowe he
oxygen consump ion in es ed bac e ia. They also
inhibi syn hesis o inne and ou e cy oplasmic
memb ane o bac e ia by pie cing he
lipopolysaccha ide laye due o he inhibi ion o e lux
pump (O hman e al., 2019).
I has been epo ed ha he e a e abou 400 species
o s ess ole an plan s in Pakis an. Halophy es ha e
been su i ed in ha sh clima es including d ough ,
cold, ho , and salini y (Rajpa e al., 2018).
The cos o using xenobio ic d ugs is 1.5 billion dolla s
annually (A sa e al., 2019). The objec i e o his
s udy was o assess he phy ochemicals om he
lea es o S. u icosa o hei an ibac e ial ac i i y.
Ma e ials and me hods
Sample collec ion
Fi m and ha mless lea es o sal - ole an Suaeda
u icosa we e collec ed om Kasu , an a ea loca ed
nea o Laho e, Pakis an. The lea es we e iden i ied
by a axonomis om he Depa men o Bo any,
Uni e si y o he Punjab, Laho e, Pakis an.
A e wa ds hey we e washed igo ously unde
unning ap wa e and sunbaked o wo weeks in an
incuba o a empe a u e o 50-70℃. They we e han
g inded o make a ine powde .
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Plan lea es powde use o ex ac and il a e
p epa a ion
The ex ac and il a e we e p epa ed wi h sol en s o
di e en pola i y namely me hanol ex ac , me hanol
il a e, and dime hyl sul oxide il a e.
The g inded lea es (2.5g) we e used in 25ml o he
sol en s and we e mixed p ope ly. The e ined
amalgam was le in he shake o 24 h and i was
cen i uged a 4000 pm o 25-30 min. The collec ed
supe na an was hen ans e ed in an emp y beake
(500 ml) o he sol en o e apo a e.
Collec ion o bac e ial s ains
The clinical isola es we e ob ained om The Se ices
Hospi al, Laho e. The bac e ial s ains used we e
Esche ichia coli, Klebsiella pneumoniae,
Pseudomonas ae uginosa, and Salmonella en e ica.
P epa a ion o he es cul u es
Inoculum o he pa hogenic bac e ial s ains was
p epa ed by cul u ing in L-B o h o o e nigh . The
bac e ial g ow h was egula ed wi h a hickness
equi alen o ha 0.5 op ical densi y o McFa land
s anda d. Ampicillin s ock solu ion was p epa ed as
s anda d o he de ec ion o an imic obial ac i i y.
An ibac e ial assays
Disc di usion assay
Mulle Hil on (MH) aga pla es we e p epa ed and
clinical isola es we e sp eade using co on swab.
Fil e pape discs o Wha man no 1 (0.5cm) soaked
wi h h ee ex ac s we e placed in p epa ed MH aga
pla es and incuba ed a 37℃.Diame e o zone o
inhibi ion was measu ed in cm.
Aga well di usion me hod
MH aga was looded wi h he inoculum o
loga i hmic phase samples o bac e ial isola es. Wells
o (0.8cm) we e cu in he aga wi h he help o
Pas eu pipe e and plan ex ac s o 50 µl we e
added. The pla es we e incuba ed a 37℃ o 24h. The
an imic obial ac i i y was e alua ed by measu ing he
diame e o zone o inhibi ion o med a ound he
wells.
De e mina ion o minimum inhibi o y concen a ion
(MIC)
MIC was de e mined by keeping he bac e ial s ains
in L-b o h a a u bidi y and op ical densi y o 0.5
MacFa land s anda ds. Me hanol ex ac , me hanol
il a e, and DMSO il a e we e added by inc easing
he concen a ion o each ex ac and il e om 20
µl/5ml o 100 µl/5ml o L-b o h and incuba ed o 24
h a 37℃. MIC concen a ion o ex ac and il a e
was aken ha did no show any isible g ow h.
Deduc ion o minimal bac e icidal concen a ion
(MBC)
Minimal bac e icidal concen a ion (MBC) was
de e mined wi h a se o ex ac and il a e ubes
ha ing no isible g ow h while de e mining MIC.
They we e s eaked on MH aga pla es and he pla es
we e incuba ed a 37℃ o 24 h.
The accumula ion o lea es ex ac and il a e a
which no isible g ow h was obse ed was conside ed
as minimal bac e icidal concen a ion.
Quali a i e assessmen o phy ochemicals
The ex ac and il a es we e es ed o he exis ence
o alkaloids, la onoids, and phenol using quali a i e
phy ochemical assessmen .
Remo al o alkaloid om plan lea es
Heal hy lea es o S. u icosa (2.5g) we e g inded
wi h 10% ace ic acid in e hanol (18-20ml)by using
mo a and pes le and incuba ed in a shady place o
4h. A e incuba ion, he ex ac was il e ed and
condensed o 20min in a boiling wa e ba h.
Ammonia (25%) was added un il p ecipi a ion and
hen cen i uged a 2500 pm o 10 min. The
emnan was washed wi h 1% NH4OH and il e ed.
The su plus was hen weighed, dissol ed in e hanol,
and s o ed a 4℃.
Sepa a ion o phenol om lea es
One g am (1g) ine powde o lea es was aken in a
conical lask and 20 ml o e hanol (80%) e hanol was
added o i . The lask was co on plugged and placed
in a boiling wa e ba h o 15 min wi h equen
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swing. I was hen cen i uged and supe na an was
collec ed.
Fla onoid ex ac ion
Hal o he supe na an was ans e ed o a 50ml
clean unnel con aining pe oleum e he (40-60℃).
Fo ma ion o an aqueous laye ep esen s la onoid
ex ac .
These sepa a ed phy ochemical ac ions om lea es
we e hen u he es ed o hei an ibac e ial ac ion.
Resul s
Zone o inhibi ion was eco ded maximum wi h
me hanolic ex ac and DMSO il a e while he
il a ed me hanol ex ac showed he leas
an ibac e ial ac i i y wi h disc and aga well di usion
p o ocols. Me hanolic lea es ex ac exhibi ed
maximum zone o inhibi ion i.e. 2.5 and 1.4cm agains
E. coli and bo h P. ae uginosa and K. pneumonia,
espec i ely (Fig. 1A). The DMSO lea es il a e was
mo e e icacious agains P. ae uginosa and S.
en e ica (Fig.1B).
Fig. 1. (A) An ibac e ial ac i i ies o me hanol ex ac , me hanol il a e, and dime hyl sul oxide il a e; (B)
An ibac e ial ac i i y o S. u icosa lea es ex ac by using Disc di usion me hod agains clinical bac e ial s ains
i.e. P. ae uginosa (a) and S. en e ica (b).
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The phy ochemical compounds i.e. alkaloids,
la onoids, and phenols ha e been de ec ed o hei
an ibac e ial ac i i y by using aga well and disc
di usion me hods (Fig. 2). The phenolic ac ion om
he lea es o S. u icosa showed 2, 2.2, 2.6, and 4cm
zone o inhibi ion espec i ely agains E. coli, K.
pneumoniae, P. ae uginosa, and S. en e ica (Fig. 2).
Fig. 2. (A) An ibac e ial ac i i ies o alkaloid, phenolic, and la onoid ac ion; (B) An ibac e ial ac i i y o plan
lea es ex ac by using Disc and Aga well di usion me hod agains P. ae uginosa (a) and S. en e ica (b) and (c).
The an imic obial ac i i y o phenolic ac ion was
g ea e as compa ed o he con ol i.e. ampicillin
using disc and aga well di usion me hod. The zone
o inhibi ion wi h ampicillin eco ded o E. coli, K.
pneumoniae, P. ae uginosa and S. en e ica was 1.9,
1.6, 2.6, and 1.9cm, espec i ely (Fig.4A,B). Alkaloid
ac ion was po en agains S. en e ica by gi ing a
zone o inhibi ion o 1.5cm as compa ed o he con ol
i.e. 1cm (Fig. 5A,B). The la onoid ac ion was po en
agains bac e ial s ains by showing zone o inhibi ion
o 2.3cm bo h o S. en e ica and E. coli and 2.1cm
bo h o K. pneumoniae, and P. ae uginosa (Fig. 2)
which we e g ea e han con ol which showed 0.9,
1.2, 0.5, and 2.2cm zone o inhibi ion agains K.
pneumoniae, P. ae uginosa, S. en e ica, and E. coli
(Fig. 6A,B).
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Fig. 3. (A) Minimum inhibi o y concen a ion o S. u icosa lea es ex ac agains clinical bac e ial s ains a a
concen a ion whe e no isible g ow h was obse ed; (B) G ow h suscep ibili y o K. pneumoniae (a), P.
ae uginosa (b), and E. coli (c) wi h la onoid and alkaloid o plan lea es ex ac o S. u icosa.
MIC was de e mined using he ex ac s agains E.
coli, P. ae uginosa, and K. pneumoniae and me hanol
ex ac MIC agains E. coli was wi hin ange o 60
µl/5ml while alkaloid ac ion MIC agains P.
ae uginosa was 80 µl/5ml (Fig. 3A). MBC was
pe o med wi h he plan ex ac s on clinical bac e ial
isola es and no isible g ow h was obse ed on MH
aga pla es (Fig. 3B).
Discussion
An imic obial esis ance has been eme ging a an
ala ming due o misuse and o e use o d ugs ha
a o he selec ion o highly modi ied o ganisms
hence inc easing he cos o heal h ca e. New
esis ance mechanisms ha e been epo ed globally in
mic oo ganisms including bac e ia i.e. E. coli
esis ance has been epo ed in many pa s o he
wo ld whe e he ea men wi h luo oquinolone
an ibio ic is now ine ec i e. Wo ld heal h
o ganiza ion has now been aking se ious measu e by
p o iding echnical ein o cemen o help coun ies
de elop hei na ional ac ion plan ha migh be
ui ul enough o comba his global conce n in nea
u u e (To nimbene e al., 2018).
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2020
Fig. 4. (A) An ibac e ial ac i i y o phenolic ex ac om lea es o S. u icosa by using Disc and Aga well
di usion me hod. (B) An ibac e ial ac i i y o phenolic ex ac om lea es o S. u icosa by using Disc and Aga
well di usion me hod.
In one o he p e ious s udies, me hanolic ex ac
om he lea es o T ian hema po ulacas um has
been used o assess i s ac i i y agains E. coli and P.
ae uginosa. The maximum zone o inhibi ion
eco ded was 1.6 and 1.3cm, espec i ely (Falade e
al., 2019). The e a e se e al epo s in he li e a u e
ha speci y he an ibac e ial po en ial o he plan s.
Cinnamomum cassia, also called Chinese cassia, is an
e e g een ea belongs o he amily named as
Lu aceae (Liang e al., 2019). The me hanol lea es
ex ac o C. cassia has been epo ed o inhibi he
g ow h o E. coli wi h a maximum zone o inhibi ion
o 1.8cm by using a disc di usion me hod (Chaudha y
e al., 2019).The size o inhibi o y zone sugges s
signi ican an ibac e ial ac i i y o he L. cama a
lea es ex ac belonging o he amily o Ve benaceae
was in es iga ed p e iously agains S. en e ica, P.
ae uginosa, and K. pneumoniae.
The me hanol ex ac had an ibac e ial ac i i y
agains S. en e ica and P. ae uginosa gi ing a zone o
inhibi ion o 2.1 and 2.0cm, espec i ely while he
ex ac was ailed o i s ac i i y agains K.
pneumoniae (Delgado-Al ami ano e al., 2019).
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Fig. 5. (A) An ibac e ial ac i i y o alkaloid ex ac along wi h s anda d by using Aga well di usion agains
bac e ial s ains. (B) An ibac e ial ac i i y o alkaloid ex ac ob ained om S. u icosa in compa ison wi h
s anda d by using Aga well di usion me hod agains S. en e ica (a) and E. coli (b).
One p e ious s udy desc ibed 4 medicinal plan s
sc eened o hei an imic obial p ope ies. The plan
subs ance used was he lea es o Pipe guineense,
Cong onema la i olium, Ocium g a issium and he
ipe ui s o Xylopia ae hiopiea. The phy ochemical
sc eening showed he p esence o alkaloids, annins,
glycosides and saponens while la onoids and phenols
we e absen (Amadi, 2018). Plan s o genus
Cle odend on belong o he amily Ve benaceae and
ha e been b oadly used o managing a ious
diseases. Quali a i e phy ochemical examina ion o
his plan con i ms he p esence o di e en
phy ochemicals i.e. s e ols, e penoids, alkaloids,
ca bohyd a es, annins, and glycosides in i s
me hanolic ex ac whe eas la onoids, phenols, and
saponins we e absen . Simila ly, he me hanolic
ex ac o lea es o Phyllan us ama us was epo ed
o he p esence o la onoids and alkaloids absence
o phenols (Malayaman e al., 2019).
Me hanol ex ac s usually show mo e inhibi ion as
compa ed o he o he ex ac s ob ained om plan s.
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2020
In a p e ious s udy, he phenolic ac ion om lea es
o T. po ulacas um shows he maximum zone o
inhibi ion wi h E. coli and P. ae uginosa had been
eco ded 0.4 and 0.2cm, espec i ely (Yamaki e al.,
2016) while he la onoid ac ion o T.
po ulacas um was po en agains P. ae uginosa
and E. coli by gi ing a maximum zone o inhibi ion
2.1 and 2.3cm, espec i ely (Gee halakshmi e al.,
2018).
Fig. 6. (A) An ibac e ial ac i i y o la onoid ex ac ob ained om lea es o S. u icosa by using Disc-Aga (F1)
and Well di usion me hod (F2). (B) An ibac e ial ac i i y o la onoid ex ac by using Aga well-Disc di usion
me hod agains P. ae uginosa and E. coli.
MIC alues in p e iously epo ed s udies we e ound
highe ha is 150-200 µl/ml wi h he xe ophy es
including Calligonum polygonides, Peganum
ha mala, and Rosa bu ononii agains E. coli (Khan e
al., 2018).C. cassia me hanolic ex ac depic s MIC
agains E. coli a he highes alue o 2640mg/l
(Mos a a e al., 2018). The MIC o me hanol ex ac
o L. cama a agains E. coli has been eco ded wi hin
he ange o 5-8mg/ml (Oduola e al., 2018). The
me hanol ex ac om T. po ulacas um showed
1.25 mg/ml MIC agains E. coli (Abd El-Gawad e al.,
2016).
Conclusion
The p esen in es iga ion clea ly demons a es ha
he me hanol lea es ex ac o S. u icosa has a b oad
