Co esponding au ho : Fouad Razzaq Al-Bu ki
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An i ungal ac i i y and syne gis ic po en ial o Achillea san olina e hanolic ex ac
and i s bioac i e cons i uen s agains ichophy on men ag ophy es
Wa aa Nase Radhi and Fouad Razzaq Al-Bu ki *
Jabi Ibn Hayyan Uni e si y o Medical and Pha maceu ical Sciences- College o Pha macy- I aq.
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
Publica ion his o y: Recei ed on 10 Augus 2025; e ised on 25 Sep embe 2025; accep ed on 27 Sep embe 2025
A icle DOI: h ps://doi.o g/10.30574/gscbps.2025.32.3.0364
Abs ac
Chemical analysis esul s o he alcoholic ex ac o Achillea san olina e ealed he p esence o 33 bioac i e compounds.
The mos p ominen o hese compounds esponsible o he ex ac 's biological ac i i y we e phy ol (17.31%),
benzophenone (14.23%), scopa one (12.78%), he 3-me hyl-3-bu en-1-ol de i a i e (13.29%), and i hiocyanu ic acid
(11.74%). MIC and MFC es s demons a ed he ex ac 's e ec i eness agains T ichophy on men ag ophy es, wi h an
MIC o 8 ± 0 mg/mL and an MFC o 9 ± 0 mg/mL, compa ed o he s anda d lucy oconazole (MIC = 16 µg/mL and MFC
= 32 µg/mL). The esul s showed ha he e hyl ac ion (Teac) eco ded he highes an i ungal ac i i y, wi h an MIC o
2.0 ± 0.1 mg/milk MFC = 3.0 ± 0.3 mg/mL when he ex ac was sepa a ed in o o ganic ac ions, while he aqueous
ac ions we e less e ec i e (MIC = 10 mg/mL, MFC >10 mg/mL). S onge ac i i y was obse ed o he isola ed
compounds scopa one and phy ol a lowe concen a ions, wi h a syne gis ic e ec when mixed in a 1:1 a io (MIC =
150 ± 15 µg/mL, MFC = 300 ± 15 µg/mL).
The esul s o he syne gis ic expe imen be ween luliconazole and he c ude ex ac indica ed a signi ican dec ease in
bo h MIC alues ( om 7 mg/mL o 3 mg/mL o he ex ac and 16 µg/mL o 6 µg/mL o he d ug), wi h an FIC index
o 0.50, indica ing a syne gis ic e ec be ween hem.
The esul s o Time-kill assay showed ha he e hyl ac ions a 2×MIC educed he mic obial densi y o less han 1.0
log CFU/mL a e 24 hou s, con i ming a ungicidal e ec , while he c ude ex ac eco ded a dec ease o 3.8 log CFU/mL.
Cy o oxici y assay showed ha he isola ed compounds had lowe oxici y and g ea e selec i i y han he c ude ex ac s
and semi-pu e ac ions. This makes hem sa e ea men op ions.
These esul s con i m he s ong po en ial o Achillea san olina as a na u al sou ce o an i ungals. I could be de eloped
in o addi ional ea men op ions, especially when used alongside adi ional d ugs.
Keywo ds: A. San olina; T. Men ag ophy es; MIC; MFC; Cy o oxici y
1. In oduc ion
Fungal skin in ec ions, including hose caused by T. men ag ophy es, a e a g owing conce n due o esis ance o many
con en ional ea men s such as luconazole, p omp ing esea che s o look o powe ul na u al an i ungal al e na i es
(Kuma e al., 2020). Annually, mo e han a billion people a ound he wo ld su e om ungal in ec ions, and ungal
pa hogens a y in hei pa hogenesis and can cause in ec ions anging om mild o li e- h ea ening (Denning, 2024).
Only a ew ypes o an i ungal d ugs a e a ailable o ea ing ungal in ec ions because hey a e e y simila o human
cells (Houšť e al., 2020).
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
247
T ea ing hese in ec ions is becoming mo e di icul because o he ising esis ance o he d ugs we ha e now (Vi iello
e al., 2023), This ield needs a lo o wo k o igh d ug- esis an ungal in ec ions. Seconda y compounds om na u al
sou ces, like e penes and couma ins, show p omise o c ea ing an i ungal ea men s wi h di e en modes o ac ion.
Fo ins ance, phy ol, which comes om plan chlo ophyll, is a long-chain e pene ha has shown an i ungal e ec s
h ough a ious mechanisms. Mos impo an ly, i damages ungal cell memb anes by causing a loss o po assium and
inac i a ing ce ain enzymes ha a e c ucial o mic oo ganism me abolism (Lima e al., 2020). Nume ous s udies ha e
shown ha na u al couma ins, like scopa one, ha e an imic obial and an i-in lamma o y p ope ies and signi ican
an ioxidan ac i i y (Ghosh e al., 2023; Wi aicenis e al., 2014).
Some s udies sugges ha na u al chemical compounds, when used wi h s anda d d ugs, can c ea e be e e ec s
agains ungi. Plan ex ac s a e able o inc ease he pe meabili y o bac e ial memb anes, inac i a e e lux pumps, and
modi y he ac i i y o p o ease enzymes, hus enhancing he e ec i eness o con en ional d ugs (Zia e al., 2025; Hosee
e al., 2025).
O e all, he esul s o nume ous s udies indica e ha bioac i e compounds such as phy ol and Couma ins especially
when used in mul iple o mula ions o ia imp o ed deli e y sys ems ha e a p omising po en ial o applica ion in he
de elopmen o an i ungal he apies, ei he independen ly o in combina ion wi h s anda d d ugs. These mechanisms
include dis up ing ungal memb anes, in e e ing wi h i al cellula p ocesses, and exe ing syne gis ic e ec s ha
enhance he e icacy o con en ional ea men s (Zai e al., 2025; Ghosh e al., 2023; Xu e al., 2023).
This s udy aimed o assess he an i ungal po en ial o (Achillea san olina L.) ex ac in ela ion o i s key bioac i e
componen s, phy ool, scopa one, and benzophenone.
2. Ma e ials and me hods
2.1. Alcoholic Ex ac ion
The s eps o ex ac ion ollowed he me hod desc ibed by Ha bo ne in 1948. We used 75% e hanol as he sol en . This
was ollowed by mace a ion, il e ing, and e apo a ion unde educed p essu e o ob ain a concen a ed ex ac o he
ac i e compounds om he lea es o A. san olin.
2.2. Phy ochemical Analysis Using Gas Ch oma og aphy
Chemical analyses es ima ed he ac i e componen con en in Achillea san olin lea es. This was done using a gas
ch oma og aphy-mass spec ome e (GC-MS) (Agilen 5977 A MSD, USA), simila o me hods used in o he s udies (da
Sil a e al., 2023; de Oli ei a e al., 2020). The Mass Hun e GC/MS Acquisi ion and Mass Hun e quali a i e so wa e
we e used. The ins umen is se o ion sou ce empe a u e 230 ℃, quad upole empe a u e 150 ℃, in e ace
empe a u e 290℃ (MSD ansmission line), s a ime 4 minu es and inish ime 35-40 minu es.
2.3. De e mina ion o MFC and MIC
The an i ungal ac i i y o he alcoholic ex ac agains T ichophy on men ag ophy es was es ed using he B o h
mic odilu ion me hod in liquid media acco ding o CLSI guidelines, 2012.
G adien concen a ions o he plan ex ac (2 o 16 mg/mL) we e p epa ed in 96-well pla es. Then, a s anda d ungal
suspension (1×10⁵ CFU/mL) was added. The pla es we e kep a 28°C o 72 hou s. The minimum inhibi o y
concen a ion (MIC) was ound o be he lowes concen a ion ha comple ely s opped ungal g ow h.
The minimum le hal concen a ion (MFC) was de e mined by ans e ing he con en s o g ow h- ee wells o esh
solid media and moni o ing he appea ance o absence o g ow h a e 72 hou s. The MFC was iden i ied as he lowes
concen a ion ha comple ely s opped ungal g ow h. (Espinel-Ing o , 2001).
2.4. An i ungal ac i i y o he ex ac a e ac iona ion
The c ude ex ac was ac iona ed in o se e al ac ions using di e en sol en s (n-hexane, dichlo ome hane, e hyl
ace a e, n-bu anol, and wa e ). Thei an i ungal ac i i y agains T ichophy on men ag ophy es was e alua ed using a
se ial dilu ion me hod in liquid media o de e mine he MIC and MFC, acco ding o (Sha ma e al., 2020).
The esul s showed signi ican a ia ion in an i ungal ac i i y depending on he sol en ype. The c ude ex ac eco ded
an MIC o 7.0 mg/mL and an MFC o 8.0 mg/mL, while he o ganic ac ions showed s onge ac i i y. The e hyl ace a e
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
248
ac ion had he highes ac i i y (MIC = 2.0 ± 0.1 mg/mL, MFC = 3.0 ± 0.3 mg/mL), indica ing a high concen a ion o
ac i e compounds. The o he ac ions we e ai ly e ec i e. Howe e , he aqueous agg ega e had lowe e ec i eness,
wi h a minimum inhibi o y concen a ion (MIC) o 10 mg/mL and a minimum ungicidal concen a ion (MFC) g ea e
han 10 mg/mL.
2.5. Syne gis ic In e ac ion be ween he C ude Ex ac and Fluconazole
The combined e ec o he c ude ex ac and luconazole was es ed using a Checke boa d mic odilu ion assay. This
was done o ind he minimum inhibi o y concen a ion (MIC) and ac ional inhibi o y concen a ion (FIC) alues, as
ou lined by Odds in 2003. G adien mix u es o he ex ac and luconazole we e p epa ed in 96-well pla es. The ungal
suspensions we e incuba ed o 72 hou s a 28°C. The F ac ional Inhibi o y Concen a ion (FIC) o each agen was
calcula ed o de e mine he ype o in e ac ion: syne gis ic, addi i e, o an agonis ic.
2.6. Analysis o he E ec o he Ex ac on he Time-Kill Assay and His o oxici y E alua ion
The s udy examined how he c ude ex ac and e hyl ace a e ac ion a ec ed he g ow h o T ichophy on
men ag ophy es h ough a Time-Kill assay, based on he wo k o Bibi e al., 2023. The esea che s incuba ed he ungus
wi h a ious concen a ions o he ex ac and ac ion o 24 hou s. They measu ed cell densi y a speci ic in e als: 0,
4, 8, 12, and 24 hou s. This helped hem unde s and how concen a ion and ime impac ed he inhibi ion o killing o
he ungus. The s udy also assessed he his o oxici y o he ex ac , ac ion, and isola ed compounds (Scopa one and
Phy ol) on HaCaT and NHDF cells. This helped de e mine he IC50 alues and he selec i i y index (SI).
3. Resul s and discussion
3.1. Analysis o he Chemical Componen s o Achillea san olina Ex ac
The esul s o able (1) indica e he p esence o 33 bioac i e compounds a di e en concen a ions a e analyzing he
alcoholic plan ex ac wi h GC-MS. Some compounds we e mo e abundan han o he s. The mos signi ican ones we e
phy ol (17.31%), benzophenone (14.23%), 3-me hyl-3-bu en-1-ol, TMS de i a i e (13.29%), i hiocyanu ic acid
(11.74%), and scopa one (12.78%), which is pa o he couma in g oup. These compounds a e among he mos
impo an ac o s esponsible o he ex ac 's biological ac i i y, enhancing i s syne gis ic and complemen a y e ec .
Recen li e a u e indica es ha Phy ol possesses an i ungal and an ibac e ial p ope ies, demons a ing i s abili y o
inhibi he g ow h o Candida spp., and is mo e e ec i e han luconazole in some cases when used in nanoca ie
sys ems (de Oli ei a e al., 2020). O he s udies esul s ha e also shown i s s ong e ec i eness as a su ace disin ec an .
The MIC50 agains C. albicans and A. nige is abou 62.5 µg/mL (Ehemj, 2020). Scopa one is a plan couma in ecognized
o i s wide ange o ac i i y, which includes an imic obial, an i-in lamma o y, an i ib o ic, and an ioxidan e ec s
(Zhang e al., 2024). I s an i ungal p ope ies ha e been p o en in ci us peel ex ac agains ungi like T ichophy on
men ag ophy es and Mic ospo um canis (Shao e al., 2007).
Benzophenone de i a i es ha e also shown an i ungal and an ibac e ial ac i i y. New de i a i es ha e been de eloped
wi h high e ec i eness agains se e al s ains (Chen e al., 2019; da Sil a e al., 2023). Benzophenones ex ac ed om
B azilian p opolis also demons a e s ong ac i i y agains Candida s ains (F ei es e al., 2019).
Table 1 Phy ochemical componen s iden i ied in ex ac o A. san olina
TIC% A ea
Compound name
RT
Peak
0.202603
2-Amino-2-me hyl-1,3-p opanediol
4.392
1
0.268444
Py azine, me hyl-
5.946
2
0.21695
Glycine, N-me hyl-N-me hoxyca bonyl-, nonyl es e
6.31
3
0.175256
Oxime-, me hoxy-phenyl-_
8.433
4
0.483472
4-Me hoxybenzyl me cap an, S- ime hylace yl-
10.083
5
12.786964
Scopa one
10.419
6
17.315668
Phy ol
10.528
7
0.164167
l-Alanine, N-me hoxyca bonyl-, idecyl es e
12.507
8
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
249
1.31587
1-Me hyl-5- luo ou acil
12.596
9
0.171991
2-(E)-Hexen-1-ol, (4S)-4-amino-5-me hyl-
13.62
10
9.272591
Benzo hiazole
13.849
11
0.215958
Conhyd in
14.161
12
0.211279
. alpha. -Ionone
14.588
13
0.422003
1,2-Benzenediol, O-(pi aloyl)-
14.966
14
0.504923
2-Me hoxy-4- inylphenol
15.093
15
0.186477
Benzenee hanol, 3-me hoxy-
15.145
16
0.335135
5-E hoxy-3,4-dihyd o-2H-py ole-2-ca boxylic acid,
16.845
17
0.154845
P opanoyl b omide, 2-b omo-2-me hyl-
17.218
18
0.168648
6,10,14-T ime hyl-2-pen adecanol, TMS de i a i e
17.305
19
10.57411
E hanone, 1-(3-hyd oxy-4-me hoxyphenyl)-
17.415
20
0.172997
2-Cyclohexen-3-ol-1-one, 2-[1-iminoe hyl]-
17.497
21
1.001355
E hyl 4-oxo-2-phenylpen anoa e
17.632
22
0.478441
2-Hyd oxy-1-(1'-py olidiyl)-1-bu en-3-one
17.741
23
0.153801
Amino(4-me hylphenyl) ace ic acid, N, N-dime hyl-,
17.968
24
0.162972
Die hyl Ph hala e
18.632
25
14.23555
Benzophenone
19.115
26
0.65311
. be a. -l-A abinopy anoside, me hyl
19.179
27
0.974071
P opionic acid, 4-hyd oxy-3-hexyl es e
19.2
28
1.100459
Bu anedini ile, 2,3-die hyl-2,3-diphenyl-
19.235
29
0.186162
Pipe idine, 4-me hyl-1-[3-me hyl-1-oxo-2-bu enyl]-
19.295
30
0.696961
4-E hoxy-2-(me hylamino) opone
19.35
31
13.29110
3-Me hyl-3-bu en-1-ol, TMS de i a i e
19.539
32
11.74867
T i hiocyanu ic acid
19.548
33
3.2. Plan ex ac : Resul s o MIC and MFC
The esul s o he MIC and MFC assay showed ha he alcoholic ex ac e ec i ely ough agains he de ma ophy e
T ichophy on men ag ophy es. This is shown in ( ables 2, 3, and 4).
The MIC es o he ex ac ( able 2) showed ha a concen a ion o 8 mg/mL was su icien o comple ely inhibi ungal
g ow h (MIC), wi h no g ow h obse ed in h ee independen eplica es, while lowe concen a ions esul ed in clea
g ow h. The MFC es esul s showed ha a concen a ion o 9 mg/mL was su icien o comple ely kill he ungus upon
cul u e, indica ing ha he ex ac possesses ungicidal ac i i y a ela i ely low concen a ions compa ed o he c ude
ex ac .
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
250
Table 2 G ow h o wells in MIC es o ex ac (±SD o 3 eplica es)
Final concen a ion (mg/mL)
Replica e 1
Replica e 2
Replica e 3
Mean ± SD
Case
10
0
0
0
0 ± 0
No g ow h
8
0
0
0
0 ± 0
MIC (No g ow h)
6
1
1
1
1 ± 0
G ow h
4
1
1
1
1 ± 0
G ow h
2
1
1
1
1 ± 0
G ow h
The esul s in able 3 also e ealed a s ong e icacy o luconazole a much lowe concen a ions (MIC = 16 µg/ml and
MFC = 32 µg/ml). Howe e , he e icacy o he ex ac a he speci ied concen a ions demons a es compa able po ency
when used as a na u al ex ac ich in ac i e compounds, highligh ing i s po en ial as a na u al an i ungal candida e .
Table 3 G ow h o wells in he MIC es o Fluconazole
Final concen a ion
Replica e 1
Replica e 2
Replica e 3
Mean ± SD
Case
64 µg/mL
0
0
0
0 ± 0
No g ow h
32 µg/mL
0
0
0
0 ± 0
MFC
16 µg/mL
0
0
0
0 ± 0
MIC
8 µg/mL
1
1
1
1 ± 0
G ow h
The esul s o able (4) and Figu e (1) indica e he di e ence be ween he ex ac and he s anda d d ug, highligh ing
he abili y o he ex ac o inhibi he g ow h o T. men ag ophy es a ela i ely low concen a ions o he c ude ex ac
(MIC = 8 mg/mL, MFC = 9 mg/mL). The an i ungal ac i i y comes om he ac i e compounds ound in he ex ac ,
including Phy ol, Scopa one, and Benzophenone. P e ious s udies ha e shown ha hese compounds ha e an i ungal
p ope ies.
Table 4 Summa y o MIC and MFC ± SD
T ea men
MIC (Mean ± SD)
MFC (Mean ± SD)
Achillea san olina ex ac (c ude)
8 ± 0 mg/mL
9 ± 0 mg/mL
Fluconazole (s anda d)
16 ± 0 µg/mL
32 ± 0 µg/mL
These esul s suppo he idea ha plan ex ac could be an e ec i e na u al emedy agains de ma ophy es. I has he
po en ial o be de eloped in o an i ungal d ugs o cosme ic p oduc s wi h mo e esea ch and de elopmen . This aligns
wi h ea lie s udies ha show he an i ungal p ope ies o plan ex ac s ich in couma ins and phenols (de Oli ei a e
al., 2020; Shao e al., 2007).
3.3. An i ungal Ac i i y o Ex ac F ac ions
The esul s o sepa a ing he c ude ex ac in o se e al ac ions showed a signi ican di e ence in an i ungal ac i i y
depending on he ype o sol en used. The c ude ex ac eco ded an MIC alue o 7.0 mg/mL and an MFC o 8.0 mg/mL,
while he o ganic ac ions showed s onge ac i i y. The e hyl ac ion (E OAc) had he highes ac i i y, wi h he lowes
MIC (2.0 ± 0.1 mg/mL) and MFC (3.0 ± 0.3 mg/mL), indica ing ha his ac ion is ich in biologically ac i e compounds.
As can be seen om he da a in able 5 and Figu e 2, he dichlo ome hane (DCM) ac ion showed good ac i i y, wi h a
minimum inhibi o y concen a ion (MIC) o 3.5 ± 0.4 mg/ml. The o he wo ac ions, n-hexane and n-bu anol, showed
in e media e ac i i y.
The aqueous esidues showed less e ec i eness, wi h an MIC alue o 10 mg/mL and no le hal e ec e en a high
concen a ions (MFC >10 mg/mL). These esul s e lec ha he ac i e compounds o a semipola na u e a e mainly
concen a ed in e hyl ac ins, which explains hei highe an imic obial ac i i y compa ed o o he ac ins. These
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
251
obse a ions a e consis en wi h wha was indica ed by Sha ma e al., 2020), who explained ha he e ec i eness o
plan ex ac s depends la gely on he na u e o he sol en used in he ex ac ion p ocess.
Figu e 1 An i ungal ac i i y o he plan ex ac and Fluconazole in e ms o MIC and MFC
Table 5 Resul s o ex ac ac iona ion
F ac ion (sol en )
MIC (Mean ± SD)
MFC (Mean ± SD)
Uni s
C ude ex ac (o iginal)
7.0 ± 0
8.0 ± 0
mg/mL
n-Hexane ac ion
5.0 ± 0.4
6.0 ± 0.4
mg/mL
Dichlo ome hane (DCM) ac ion
3.5 ± 0.4
4.2 ± 0.3
mg/mL
E hyl ace a e (Teac) ac ion
2.0 ± 0.1
3.0 ± 0.3
mg/mL
n-Bu anol ac ion
5.0 ± 0.4
6.0 ± 0.3
mg/mL
Aqueous esidue
10.0 ± 0.6
>10 (no kill)
mg/mL
The esul s o able (6) and Figu e (3) showed ha he isola ed compounds showed an i ungal ac i i y a lowe
concen a ions han he c ude ex ac . Scopa one had a MIC o 250 ± 10 µg/mL and an MFC o 500 ± 10 µg/mL. Phy ol
eco ded a MIC o 400 ± 30 µg/mL and an MFC o 1000 ± 40 µg/mL. The mix u e o Scopa one and Phy ol in a 1:1 a io
showed a clea d op in MIC o 150 ± 15 µg/mL, wi h an MFC o 300 ± 15 µg/mL. which indica es a possible syne gis ic
e ec be ween he wo compounds.
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252
Figu e 2 An i ungal ac i i y (MICand MFC) o ex ac ac ions
Table 6 E icacy o isola ed compounds
Compound
MIC (Mean ± SD)
MFC (Mean ± SD)
Uni s
Scopa one (pu i ied)
250 ± 10
500 ± 10
µg/mL
Phy ol (s anda d/pu i ied)
400 ± 30
1000 ± 40
µg/mL
Benzophenone de i a i e
300 ± 20
600 ± 30
µg/mL
Mix u e (Scopa one + Phy ol, 1:1 w/w)
150 ± 15
300 ± 15
µg/mL
Figu e 3 E icacy o isola ed compounds, MIC and MFC alues wi h s anda d de ia ions
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
253
3.4. Syne gis ic In e ac ion be ween C ude Ex ac and Fluconazole
The esul s o he Checke boa d es in able 7 and Figu e 4 showed ha combining he c ude ex ac wi h he an i ungal
d ug Fluconazole led o a no able dec ease in he MIC alues o bo h subs ances. The MIC alue o he c ude ex ac ell
om 7 mg/mL o 3 mg/mL when mixed wi h Fluconazole. Meanwhile, he MIC alue o Fluconazole d opped om 16
µg/mL o 6 µg/mL. When calcula ing he F ac ional Inhibi o y Concen a ion (FIC), bo h agen s had a alue o 0.50,
showing a syne gis ic e ec be ween hem. These esul s demons a e ha combining he plan ex ac wi h a
adi ional an i ungal can enhance he apeu ic e icacy and educe he concen a ion equi ed o achie e inhibi o y
ac i i y, which opens up p ospec s o he de elopmen o mo e e icien and less oxic combined he apeu ic s a egies .
These esul s a e consis en wi h wha Odds (2003) indica ed ha syne gism be ween plan ex ac s and an i ungals
may con ibu e o enhancing he he apeu ic esponse and educing he possibili y o de eloping d ug esis ance.
Table 7 Checke boa d (Ex ac + Fluconazole)
Agen
MIC alone
mg/mL
MIC in combina ion
mg/mL
F ac ion o MIC
Achillea c ude ex ac (A)
7
3
0.50
Fluconazole (B)
16 µg/mL
6 µg/mL
0.50
Figu e 4 A compa ison o he MIC alues alone and MIC in combina ion o bo h he c ude ex ac and Fluconazole
3.5. Time-Kill Assay Analysis
The da a in able (8) and Figu e (5) ep esen he Time-kill es , which shows ha ea men wi h he c ude ex ac and
e hyl ac ion (Teac) had a clea inhibi o y e ec on mic obial g ow h compa ed o he un ea ed con ol. Cell coun s in
he con ol ea men emained almos cons an a ~6.5 log CFU/mL o up o 24 hou s, while hey g adually dec eased
wi h di e en ea men s depending on concen a ion and ime. I was no ed ha he c ude ex ac a a concen a ion
o 1×MIC esul ed in a small educ ion o 5.5 log CFU/mL a e 24 hou s. In con as , he c ude ex ac a 2×MIC
demons a ed a g ea e inhibi ion o 3.8 log CFU/milk The e hyl ac ion also showed a signi ican ly highe e ec i eness.
The mic obial densi y d opped o 3.0 log CFU/mL a 2×MIC a e 24 hou s and ell below 1.0 log CFU/mL, indica ing a
bac e icidal e ec . These esul s emphasize he e ec i e ole o e hyl ac iona ion compa ed o he c ude ex ac . They
also con i m ha an ibac e ial ac i i y inc eases wi h highe concen a ion and longe exposu e ime.
GSC Biological and Pha maceu ical Sciences, 2025, 32(03), 246-257
254
Table 8 Time-kill e ec
Time
(h)
Con ol (no
d ug)
C ude ex ac
1×MIC
C ude ex ac
2×MIC
Teac ac ion
1×MIC
Teac
2×MIC
0
6.5
6.5
6.5
6.5
6.5
2
6.6
6.3
6.0
6.0
5.5
4
6.6
6.1
5.6
5.4
4.6
8
6.7
5.8
4.8
4.2
3.1
24
6.5
5.5
3.8
3.0
<1.0
The esul s om able (8) and Figu e (6) showed ha he c ude ex ac had an IC50 o 1400 ± 90 µg/mL on HaCaT cells
and 1500 ± 120 µg/mL on NHDF cells. The e hyl ac ion E OAc had an IC50 o 500 ± 50 and 750 ± 50 µg/mL. Bo h
eco ded low selec i i y alues o 0.20 and 0.25. This indica es a high ela i e oxici y when compa ed o he an i ungal
e ec i eness. In con as , he isola ed compounds Scopa one (IC50 = 1200 ± 70 and 1200 ± 80 µg/mL, SI = 4.8) and
Phy ol (IC50 >2000 µg/mL, SI = 5.0) displayed lowe oxici y and highe selec i i y, which makes hem sa e as
ea men op ions. These indings ag ee wi h Bibi e al., 2023, who no ed ha pu e plan compounds o en demons a e
be e selec i i y and lowe oxici y han c ude ex ac s o semi-pu e ac ions.
Figu e 5 Time-Kill Assay o Ex ac s, showing he ime-kill e ec o he c ude ex ac and E OAc ac ions a 1×MIC
and 2×MIC concen a ions
Table 9 Cy o oxici y (MTT assay on mammalian cells
T ea men
IC50 HaCaT (µg/mL)
IC50 NHDF (µg/mL)
Selec i i y Index (SI = IC50/MIC)
C ude ex ac
1400 ± 90
1500 ± 120
0.20
E OAc ac ion
500 ± 50
750 ± 50
0.25
Scopa one
1200 ± 70
1200 ± 80
4.8
Phy ol
>2000
>2000
5.0
