Co esponding au ho : Lambe Okwubie
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion Liscense 4.0.
E alua ion o he an imic obial and la icidal ac i i ies o n-hexane oo ex ac o
Ixo ia coccinea L. (Rubiaceae)
Lambe Okwubie 1, *, Dan John 2, Emmanuel P ince Ihechi 1, Emmanuel Ibeh Ajibo 1, Samuel Pe e Egbe 1,
Magnus Asuoye Ome ejo 1 and P ecious Minaseidiema 1
1 Depa men o Pha macognosy and Phy o he apy, Uni e si y o Po Ha cou , Ri e s s a e, Nige ia.
2 Depa men o Pha maceu ical Mic obiology and Bio echnology, Uni e si y o Po Ha cou , Ri e s s a e, Nige ia.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 302-308
Publica ion his o y: Recei ed on 29 No embe 2024; e ised on 08 Janua y 2025; accep ed on 10 Janua y 2025
A icle DOI: h ps://doi.o g/10.30574/wjbphs.2025.21.1.0010
Abs ac
The an imic obial as well as he la icidal ac i i ies o n-hexane oo ex ac o Ixo ia coccinea agains Aedes aegyp i was
e alua ed in his wo k. The ex ac ion was done using mace a ion me hod. Aga well di usion me hod was used o
assess he an imic obial ac i i y o he ex ac agains di e en clinical isola es o g am posi i e and g am-nega i e
bac e ia. The la icidal ac i i y was e alua ed acco ding o Wo ld Heal h O ganiza ion (WHO) guideline o la icidal
assay as ollow: a s ock solu ion o 10mg/ml was p epa ed o each ex ac om which i e di e en concen a ions
(1.0, 2.0, 3.0, 4.0 and 5mg/ml in 100ml) we e made and used o he es . A con ol was also p epa ed o each ex ac .
A o al o 20 la ae we e used o each o he concen a ion and mo ali y was eco ded a 24, 48 and 72 hou s. The
esul o he an imic obial assay showed ha a 100mg/ml concen a ion, he ex ac ga e an inhibi ion zone diame e
o 17±1.41mm o S aphylococcus au eus, 8±0.82mm o Bacillus spp, 2±0.00mm o S apylococcus epide midis,
4±0.58mm o Pseudomonas ae uginosa and 6±1.29mm o Esche ichia coli. The ex ac was mos ac i e agains
S aphylococcus au eus wi h an MIC o 50mg/ml. The ex ac is no ac i e agains all he ungi species used in he es .
Examina ion o he esul o la icidal assay agains Aedes aegyp i la ae showed ha he LC50 a e 24, 48 and 72
hou s a e 1.782, 1.376 and 0.686mg/ml espec i ely. A concen a ion o 3mg/ml he e was 100% mo ali y a e 72
hou s. The esul s showed ha he n-hexane oo could se e as a sou ce o bo h an ibio ic and mosqui o la icides.
Keywo ds: La icidal; An imic obial; Lxo ia coccinea; N-hexane Ex ac
1. In oduc ion
Plan s ha e long been ecognised as impo an na u al p oduc sou ces o p ese ing bo h human and animal heal h.
The e ha e been epo s sugges ing a wide a ie y o chemical compounds ound in plan s ha e signi ican he apeu ic
and p e en a i e p ope ies [1]. App oxima ely 80% o people in de eloping na ions u ilise adi ional medicines made
o subs ances de i ed om medicinal plan s [2]. Despi e he a ailabili y o se e al d ug de elopmen me hods, plan s
emain he main sou ce o na u al medicines. Because o an ibio ic esis ance, he e is enewed in e es in plan s ha
possess an ibac e ial cha ac e is ics. This esis ance may be linked o he ca eless use o comme cial medica ions o o
no ollowing p esc ip ion guidelines when using an ibio ics, such as no inishing he en i e cou se o he apy o
in ec ious in ec ions [3]. Fu he mo e, some an ibio ics ha e un a ou able side e ec s such nausea, bone ma ow
supp ession, h ombocy openic pu pu a, and ag anulocy osis, which migh cause a e diso de s o a ise [4]. This has
encou aged scien is s o look o mo e inno a i e and di e en mic obial compounds om he apeu ic plan s [3].
As a esul o he ongoing use o a i icial pes icides o con ol he Aedes aegyp i ec o , mosqui o popula ions ha e
become na u ally esis an o a ious chemical g oups.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 302-308
303
As one o he leading causes o illness and mo ali y a ound he wo ld, he e iological agen s o human i al in ec ions
sp ead by mosqui o ec o s a e o signi ican public heal h impo ance. Aedes aegyp i is conside ed a signi ican
a bo i us ca ie among mosqui o ec o s [5]. This ec o is capable o ansmi ing he pa hogens ha cause diseases
such as dengue, Zika, chikungunya, and yellow e e . Chemical insec icides like ca bama es, py e h oids,
o ganochlo ines, o ganophospha es, and syn he ic la icides a e used ex ensi ely o con ol hese mosqui oes. In
addi ion o ha ming he ecosys em, he ongoing use o hese a i icial chemicals has led o he ise o esis an
popula ions o pa hogens and insec s [6,7].
The mosqui o, Aedes aegyp i, is ound h oughou Ame ica and opical coun ies, wi h he excep ion o Canada and
Chile. Addi ionally, i is he ec o o a numbe o human- ansmi ed illnesses, such as dengue, mala ia, and, mo e
ecen ly, zika and chikungunya. In Ecuado , ec o -bo ne illnesses ank second in e ms o mo bidi y, wi h dengue and
chikungunya leading he pack.
The lowe ing plan Ixo a coccinea, o en called jungle ge anium, lame o he woods, jungle lame, o pendkuli, belongs
o he Rubiaceae amily. This ubiqui ous lowe ing sh ub is indigenous o Bangladesh, S i Lanka, and Sou he n India. I
is now one o he mos widely used blooming sh ubs in landscapes and ga dens in Sou h Flo ida [8]. Ayu eda uses his
comme cially signi ican medicinal he b. E e y po ion, including he lowe , lea es, and oo , is used o make di e en
he apeu ic emedies o condi ions like diabe es and skin diseases.
The objec i e o his wo k is o e alua e he an imic obial ac i i y o he n-hexane oo ex ac o Ixo a coccinea using
s anda d me hods. The la icidal ac i i y o he same ex ac will also be e alua ed agains Aedes Aegyp i la ae by
s anda d guideline o la icidal assay
2. Ma e ial and me hods
Ro a y e apo a o (Labscience, England), a wa e ba h (Techmel and Techmel, USA), an analy ical weighing scale, glass
unnels, beake s, c ucibles, a measu ing cylinde , a desicca o , ace one, wa e , and DMSO sol en e c. a e a ew o he
ma e ials u ilized in his wo k.
2.1. Plan collec ion
Ixo a coccinea Linn. (Rubiaceae) oo s we e collec ed om Ri e s s a e, Nige ia on he mon h o July. i was iden i ied
by D . Sulieman Mikailu o Depa men o Pha macognosy and Phy o he apy, Uni e si y o Po Ha cou and deposi ed
a he same Depa men He ba ium, wi h he he ba ium ouche numbe UPHRO615.
2.2. Plan P epa a ion
The collec ed plan oo s we e cu in o smalle pieces, subjec ed o ho ough washing, and ai -d ied unde a shade un il
well d ied. Following he d ying pe iod, he oo s we e inely pul e ized and secu ely s o ed in an ai igh con aine o
subsequen u iliza ion.
2.3. Ex ac ion o Plan Ma e ials
The plan ma e ial ex ac ion p ocess was done using he mace a ion me hod. This was done wi h he measu emen o
200g o he sample, which was subsequen ly placed in o a mace a ion ja . Su icien olume o analy ical-g ade N-
Hexane sol en was added o he sample wi hin he ja and co e ed. Tho ough mixing was achie ed h ough shaking.
The esul ing mix u e was le o mace a e o a 24-hou du a ion, wi h in e mi en shaking o acili a e enhanced
ex ac ion. Subsequen ly, he mix u e was subjec ed o il a ion. This en i e p ocedu e was epea ed using he same
ma c o ensu e exhaus i e ex ac ion. The esul ing ex ac was concen a ed using a o a y e apo a o and inally d ied
in a wa e ba h a a empe a u e 45 ℃.
2.4. Phy ochemical Sc eening
The n-hexane ex ac o he pul e ized plan oo s was subjec ed o phy ochemical e alua ion in acco dance wi h he
me hod by Ha bo ne [9].
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2.5. An imic obial Assay
2.5.1. S anda diza ion o Cul u e
A e being asep ically injec ed in o egula saline, he mic obes ha we e isola ed we e cul u ed o 24 hou s a 37 °C.
Nex , he 0.5 MaC-Fa land u bidi y s anda d was used o compa ison.
An imic obial Suscep ibili y Tes (AST) o n-hexane ex ac o Ixo a coccinea on clinical isola es Using Aga Di usion
Me hod
Co on wool and an isep ic we e used o clean he wo k op. Fo each es o ganism, s e ile Pe i pla es we e ma ked in
iplica e. The p epa ed Nu ien Aga pou in he uni e sal bo le was illed wi h 0.1 ml o each mic obe asep ically and
well mixed. A e ha , he mix u e was ans e ed o he app op ia e Pe i dish and le o ha den on he wo kbench.
A s e ile co k bo e wi h a 6 mm diame e was used o ex ac an aga disc om he aga laye once he aga had se on
he pe i dish. This allowed each aga pla e o ha e a well. The N-hexane s ock concen a ions (100 mg/ml) o Ixo a
coccinea ex ac we e ma ked on he wells. A s e ile Pas eu 's pipe e was used o ca e ully ans e 0.1 ml o each o
he p epa ed s ock concen a ions in o he well. The well was hen se on he wo kbench o 15 minu es o allow o
op imal di usion. Fo wen y- ou hou s, he pla es (Pe i dishes) we e incuba ed a 37 °C. The same p ocedu e was
epea ed using Sabou aud dex ose aga (SDA) which was inocula ed wi h he ungi and incuba ed a ambien
empe a u e o 2- 7 days. Using a me e ule, he diame e o he esul an Zones o inhibi ion was measu ed in
millime es (mm) ia he pla e bases and esul s eco ded.
De e mina ion o he Minimum Inhibi o y Concen a ion (MIC)
Co on wool and an isep ic we e used o clean he wo k op. Fo each es o ganism, s e ile Pe i pla es we e ma ked in
iplica e. The aga pou s we e inocula ed wi h 0.1ml o s anda dized bac e ia cul u e and mixed well. The inocula ed
aga was pou ed in o he labelled pe i dishes and le o solidi y on he wo kbench. A e solidi ica ion o he aga , i e
(5) wells we e bo ed using a s e ile co k bo e . These wells we e labelled o accommoda e i e (5) concen a ions o
he ex ac , he concen a ions we e 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, and 3.125mg/ml. Using a s e ile
pas eu ’s pipe e, 0.1ml o each concen a ion o he ex ac was in oduced in o he co esponding wells. The pla es
we e le on he wo kbench 15 minu es o p ope di usion and i was he incuba ed upside down a 37oC o 24 hou s.
A e incuba ion he pla es we e obse ed o p esence o zones o inhibi ion.
2.6. La icidal Bioassay
The Aedes aegyp i egg o he assay was ob ained om he Na ional ai bo ne i al esea ch cen e Enugu, Nige ia. I was
ha ch and he la ae g ow in he depa men o Pha macognosy and Phy o he apy o Uni e si y o Po Ha cou . The
La icidal bioassay was ca ied using he 3" and 4" ins a la ae o Aedes aegyp i and his was done acco ding o Wo ld
Heal h O ganiza ion [WHO, 2005] [10] s anda d me hod o la icidal assay. A s ock solu ion o 10mg/ml o he n-
hexane oo ex ac was p epa ed by dissol ing 4.5 g o he n-hexane oo ex ac in 450 ml o dis illed wa e , su icien
amoun o ace one was used o dissol e he n-hexane oo ex ac be o e adding he 450 ml o dis illed wa e and
p ope ly s i ed using a glass od, F om he s ock solu ion ha was p epa ed, se e al concen a ions was p epa ed in
iplica es (1,2,3,4 and 5mg/ml). Each o he concen a ions had a con ol con aining 100ml o dis illed wa e , 20 heal hy
la ae pu in each o he plas ic pla e used o ca y ou he assay which was un simul aneously a he same condi ions
a oom empe a u e. Mo ali y was checked o a e 24,48 and 72hou s. The dead la ae we e con i med by no
espond o s imuli o being p obed using a needle. esul s we e ca e ully obse ed and eco ded
2.7. S a is ical Analysis
The s a is ical ools ha we e used in his s udy is he Ldp line so wa e based on he s anda d me hod o p obi s by
Finney [11], o calcula e LC 50 alues o each ex ac a e he a ious ime o obse a ions which a e 24, 48 and 72
hou s.
3. Resul s
Table 1 Pe cen age yield om ex ac ion o Ixo a coccinea Linn
Weigh o plan ma e ial (g)
Sol en
Weigh o ex ac (g)
Pe cen age yield (%)
200
n-hexane
8.75
4.375
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305
Table 2 Resul o he Phy ochemical sc eening o Ixo a coccinea oo ex ac
S/No
Phy ochemical
Resul
1
An h aquinone
+
2
Alkaloids
-
3
Fla onoids
+
4
Saponin
+
5
Phenolic compounds
+
6
Ca bohyd a e
+
7
T i e penoids
+
Key: (+): - posi i e, (-): - nega i e
Table 3 Resul o he An imic obial Suscep ibili y Tes (AST) o N-Hexane ex ac o Ixo a coccinea oo
Mic oo ganism
Conc.
mg/ml
n-hexane ex ac
mean IZD (mm)
Posi i e con ol (gen amicin 2mg/ml)
mean IZD (mm)
Nega i e con ol
DMSO
G am posi i e
S aphylococcus
au eus
50
100
8 ± 0.82
17±1.41
4 ± 1.41
Nil
Bacillus spp
50
100
Nil
3 ± 0.82
3 ± 0.00
Nil
S aphylococcus
epide midis
50
100
Nil
2 ± 0.00
6 ± 1.73
Nil
G am nega i e
Pseudomonas
ae uginosa
50
100
Nil
4 ± 0.58
3 ± 0.00
Nil
Esche ichia coli
50
100
Nil
6 ± 1.29
7 ± 0.82
Nil
Klebsiella spp
50
100
Nil
Nil
9 ± 1.00
Nil
P o eus spp
50
100
Nil
Nil
8 ± 2.16
Nil
Fungi
Posi i e con ol (Fluconazole 5mg/ml)
Nega i e con ol DMSO
Candida spp
50
100
Nil
Nil
18 ±2.45
Nil
Aspe gillus spp
50
100
Nil
Nil
Nil
Nil
Penicillium spp
50
100
Nil
Nil
Nil
Nil
Values o means a e ep esen ed as mean o ± S.E.M (s anda d e o o means). Key: IZD =Inhibi ion zone diame e
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306
F om he able 3 abo e he esul o he an imic obial suscep ibili y es showed ha he ex ac is e y ac i e agains
S aphylococcus au eus mode a ely ac i e agains Pseudomonas ae uginosa and Esche ichia coli and poo ly ac i e agains
Bacillus spp and S aphylococcus Epide midis. All he ungi spps a e esis an o he ex ac .
Table 4 Minimum Inhibi o y Concen a ion (MIC) o N-Hexane ex ac o Ixo a coccinea oo
O ganisms
Concen a ion
3.125
6.25
12.5
25
50
100
S aphylococcus au eus
+
+
+
+
-
-
Bacillus spp
+
+
+
+
+
-
S aphylococcus epide midis
+
+
+
+
+
-
Pseudomonas ae uginosa
+
+
+
+
+
-
Esche ichia coli
+
+
+
+
+
-
Key: (+) g ow h, (-) inhibi ion
F om he esul o MIC in able 4 abo e, 100mg/ml is he minimum inhibi o y concen a ion o all he o ganisms wi h
he excep ion o S aphylococcus au eus which mic o 50mg/ml.
Table 5 Resul o la icidal assay o n-hexane oo ex ac o Ixo a coccinea agains Aedes aegyp i
Con. Mg/ml
24 hou s
48 hou s
72 hou s
% mo ali y
LC50
Con ol
% mo ali y
LC50
Con ol
% mo ali y
LC50
Con ol
1
18.3±19.3
1.782
0
26.7±20.1
1.376
0
70±4.1
0.686
0
2
61.7±20.9
0
81.7±13.1
0
91.7±8.5
0
3
81.7±8.5
0
86.7±2.4
0
100±0.0
0
4
81.7±4.7
0
90±8.2
0
98.3±2.4
0
5
90±0.0
0
96.7±4.7
0
100±0.0
0
Values o means a e ep esen ed as mean o ± S.E.M (s anda d e o o means)
Resul o he la icidal assay in he able 5 abo e showed ha he ex ac ga e s ong la icidal ac ion agains he Aedes
aegyp i la a wi h LC50 o 1.78mg/ml a e 24 hou s and 0.686mg/ml a e 72 hou s
4. Discussion
Examina ion o esul o pe cen age yield showed he yield o be 4.375%. he low pe cen age yield may be due o he
sol en o ex ac ion
Phy ochemical sc eening ga e he p esence o an h aquinone, la onoids, saponin, phenolic compounds, ca bohyd a es
and i e penoids. The e is absence o alkaloids. This is simila o esul ob ained by Okwubie and Ajogwu, (2021) [12]
which eco ded he p esence o hese compounds wi h addi ional p esence o alkaloids and cyanogene ic glycosides,
while Joshi e al., (2013) [13] epo ed he p esence o alkaloids, ca bohyd a es, la onoids, glycosides, annins, esins,
saponins, i e penoids and s e oids in he e hanolic oo ex ac . This may be due o di e en sol en s o ex ac ions.
One o wo o hese phy ochemicals may be esponsible o he la icidal and he an imic obial ac i i ies.
The esul o he an imic obial ac i i ies o he ex ac showed e y good ac i i y agains S aphylococcus au eus wi h
inhibi ion zone diame e o 8 ± 0.82 and 17±1.41mm o dose concen a ion o 50 and 100mg/ml espec i ely. This
ga e mo e ac i i y han he posi i e con ol o gen amycin wi h inhibi ion zone diame e o 4 ± 1.41mm. Though he e
we e ac i i ies agains Bacillus spp, S aphylococcus epide midis, Pseudomonas ae uginosa, Esche ichia coli bu hey we e
a highe concen a ion o 100mg/ml as such he ex ac s has lowe an ibac e ial ac i i ies agains hese o ganisms
when compa e wi h he s anda d, as shown on he able 3 abo e. Ma imu hu e al., (2014) [14] showed ha he
me hanolic ex ac o his plan ga e good ac i i ies agains he bac e ia used in his s udy, hough he ac i i ies end o
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307
be highe wi h he me hanolic lea , s em and lowe ex ac s om ha s udy han he hexane oo ex ac used in his
s udy. Howe e , Ma imu hu e al (2014) [14] showed ha he me hanolic ex ac s ga e good ac i i ies agains ungi
while his s udy showed ha he n-hexane oo ex ac ga e no ac i i y agains ungi. Also, Naga aj Sel a aj e al., (2011)
[15] showed ha e hanol ex ac ga e highe an ibac e ial ac i i y when compa e o lesse pola sol en s. Howe e ,
hey epo ed e y low an i ungal ac i i ies wi h all he sol en s used in hei wo k which is close o he esul on he
an i ungal ac i i y in his wo k.
Examina ion o he la icidal ac i i y showed ha he n-hexane ex ac exhibi ed a e y good la icidal ac i i y agains
Aedes aegyp i la ae. A concen a ion o 1mg/ml he e was 70±4.1 pe cen age mo ali y a e 72 hou s. The pe cen age
mo ali y inc eased o 91.7±8.5 a e 72 hou a concen a ion o 2mg/ml, a concen a ion o 3mg/ml he pe cen age
mo ali y became 100 pe cen age a e 72 hou s. The la icidal ac i i ies o his ex ac is dose and ime dependen .
This is collabo a ed by he wo k done by Okwubie and John, 2017 [16] and Nwabo e al, 2014 [17] which showed he
ac i i y is dose and concen a ion dependen .
Okwubie and Ajogwu, (2021) [12] had shown ha ace one oo ex ac o his plan has e y high ac i i y, while ex ac s
wi h high pola sol en o me hanol and wa e ga e low and e y low ac i i y espec i ely. This showed ha he
la icidal ac i i y eside wi h ex ac s om non-pola sol en s o mid pola sol en s as he esul on he use o non-
pola sol en n-hexane in his wo k showed e y good ac i i y. The e o e, he bes sol en o ex ac ion o good
la icidal ac i i y is non-pola sol en s.
5. Conclusion
This wo k has shown ha n-hexane oo ex ac o Ixo a coccinea has bo h an ibac e ial and la icidal ac i i y bu has
no an i ungal ac i i y. On he la icidal ac i i y, u he wo k could be made o isola e and cha ac e ize he compound
o agen esponsible o la icidal ac i i y.
Compliance wi h e hical s anda ds
Acknowledgmen
We acknowledge he con ibu ions o all he labo a o y echnologis in his esea ch wo k.
Disclosu e o con lic o in e es
We he au ho s do decla e ha he e is no con lic o in e es .
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