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Antifungal Activity of Jasminum sambac against Malassezia sp. and Non-Malassezia sp. Isolated from Human Skin Samples

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Malassezia sp. causes skin diseases such as pityriasis versicolor, folliculitis, and atopic dermatitis. The present study aims to evaluate the antifungal activity of J. sambac or Arabian jasmine, a flowering plant abundant in the Southeast Asia against Malassezia sp. using disc diffusion and broth microdilution method. The methanol extract and essential oil from the flowers and leaves of J. sambac were, respectively, prepared using solvent extraction and hydrodistillation process. Skin samples from individuals with dandruff were cultured on Sabouraud dextrose agar overlaid with olive oil. The fungi that grew were observed microscopically, tested with Tween assimilation test, and cultured on CHROMagar (the chromogenic media pioneer) to identify Malassezia sp. Out of 5 skin samples, only 2 Malassezia sp. isolates were identified based on morphology and their ability to assimilate Tween. The inhibition zones of methanol extract of flowers and leaves of J. sambac and essential oil of flowers showed potential for antifungal activity with inhibition zones of 11.10 ± 1.92, 12.90 ± 1.68, and 13.06 ± 0.26 mm, respectively, and minimum inhibitory concentration (MIC) values of 80 mg/mL to 160 mg/mL and 50%, respectively. In conclusion, J. sambac may be used as an alternative treatment against Malassezia-associated skin infections.
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Research Article
Antifungal Activity of Jasminum sambac against Malassezia sp.
and Non-Malassezia sp. Isolated from Human Skin Samples
Jacinta Santhanam, Farhana Nadiah Abd Ghani, and Dayang Fredalina Basri
School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia,
JalanRajaMudaAbdulAziz,50300KualaLumpur,Malaysia
Correspondence should be addressed to Dayang Fredalina Basri; dayang@ukm.edu.my
Received  July ; Revised  October ; Accepted  October ; Published  November 
Academic Editor: Simona Nardoni
Copyright ©  Jacinta Santhanam et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Malassezia sp. causes skin diseases such as pityriasis versicolor, folliculitis, and atopic dermatitis. e present study aims to evaluate
the antifungal activity of J. sambac or Arabian jasmine, a owering plant abundant in the Southeast Asia against Malassezia sp. using
disc diusion and broth microdilution method. e methanol extract and essential oil from the owers and leaves of J. sambac
were, respectively, prepared using solvent extraction and hydrodistillation process. Skin samples from individuals with dandru
were cultured on Sabouraud dextrose agar overlaid with olive oil. e fungi that grew were observed microscopically, tested with
Tween assimilation test, and cultured on CHROMagar (the chromogenic media pioneer) to identify Malassezia sp. Out of  skin
samples, only  Malassezia sp. isolates were identied based on morphology and their ability to assimilate Tween. e inhibition
zones of methanol extract of owers and leaves of J. sambac and essential oil of owers showed potential for antifungal activity
with inhibition zones of . ±., . ±., and . ±. mm, respectively, and minimum inhibitory concentration (MIC)
valuesofmg/mLtomg/mLand%,respectively.Inconclusion,J. sambac maybeusedasanalternativetreatmentagainst
Malassezia-associated skin infections.
1. Introduction
Malassezia sp. is a type of glabrous fungus which causes
infections of supercial layer of the skin. Malassezia species
are normal ora found on the skin of  billion humans but
theycanbepathogeniccausingthecommonskindisorder
which includes pityriasis versicolor, folliculitis, and atopic
dermatitis in humans []. In immunocompromised hosts,
Malassezia can also cause systemic infections. Malassezia are
lipophilic or lipid-dependent fungi requiring long chain fatty
acids, essential for the growth of most Malassezia species,
and are being supplied from the human skin lipids [].
is lipid requiring property causes the highest density of
Malassezia in the sebaceous areas such as the scalp, face, and
upper trunk and the lowest density on the hands. Being a
lipophilic yeast, therefore in vitro growth must be stimulated
by natural oils or other fatty substances. e most common
method used is to overlay Sabouraud dextrose agar with
olive oil []. Reproduction of Malassezia sp. is by budding
from a broad base present on the same cell pole (monopolar
budding) []. Prolonged use of topical antifungals such as
Itraconazole, Fluconazole, and Terbinane to treat infections
caused by Malassezia sp. has its drawbacks by causing side
eects such as burning, stinging, or redness when applied to
the skin. e main problem with oral antifungals is nausea
and dizziness on continuous use []. Other drawbacks of
commercial antifungals, whether topical or oral, are their
adverse drug interaction with other medications and high
cost in pharmacies [].
In an eort to nd an alternative from a more natural and
safer source, it is very applicable to utilize the use of natural
products. Recent study []onJasminum sambac,aowering
plant abundant locally in Southeast Asia and traditionally
used as remedies for skin problems such as acne, whitehead,
and blackhead, has proven successful. is Arabian jasmine
plant is distinctive for its owers white petals which emit a
fragranceodourandbloomthroughouttheyear.eleaves
of J. sambac contain the secoiridoid glycosides, jasminin,
quercitrin, and rutin []. e preliminary phytochemical
studyoftheantimicrobialactivityofethanoliccallusextracts
Hindawi Publishing Corporation
Journal of Mycology
Volume 2014, Article ID 359630, 7 pages
http://dx.doi.org/10.1155/2014/359630
Journal of Mycology
of jasmine focused on two species of Jasminum, J. grandi-
orum and J. sambac, showed that the plants can be used
as medicine for skin disorders []. As J. sambac possesses
antibacterialproperty,itcanbeusedtotreatacneandskin
infections. Essential oil and extracts of J. sambac have been
shown to exhibit antibacterial and antifungal properties [].
Jasmine oil has been proven to reduce skin inammation,
tones the skin by repairing skin cells by encouraging cell
growth, and increases skin elasticity [].
Jasminum sambac has also been known to inhibit the
growth of Alternaria sp.and dermatophytes[]. e callous
extracts of J. sambac were also reported to display antimicro-
bial activity against Staphylococcus albus,Proteus mirabilis,
Salmonella typhi [], and Propionibacterium acnes []. e
use of J. sambac extract can be a potential additive in skin and
hair products to prevent Malassezia sp. infection altogether
[]. e present study was therefore undertaken to conrm
the eectiveness of methanol extract and essential oil from
the leaves and owers from J. sambac against Malassezia sp.
isolated from the skin samples of individuals with dandru.
2. Materials and Methods
2.1. Plant Materials. Fresh owers and leaves of Jasminum
sambac were purchased from a nursery located in Sungai
Buloh, Selangor, Malaysia. Prior to analysis, the owers
and leaves were taken out of the freezer, thawed at room
temperature, and then washed thoroughly under running tap
water. e leaves and the owers were then spread on the tray
and allowed to dry at room temperature for several days. e
dried owers and leaves were grinded into powder using an
electrical blender. Another batch of the owers was le fresh
for hydrodistillation process to obtain the essential oil.
2.2. Extraction of Plant. Two m e thods [ ], namely, hydrodis-
tillation and methanol extraction, were employed to produce
essential oil and the crude methanol extract, respectively.
In the preparation of essential oil, about  g of powdered
leaves and  g of fresh owers were added to a  mL
round-bottom ask and lled half full with distilled water.
e  mL distilling ask was rested on the heating mantel
and was heated slowly while adding water sparingly from
the separatory funnel to prevent the plant from drying
out and burning. e distillation was stopped when the
distillate reached  mL. e distillate was extracted three
times with  mL aliquot of dichloromethane. e combined
dichloromethane layers were then extracted three times
with  mL aliquot of saturated NaCl. Magnesium sulphate
(MgSO4) was used as a drying agent to dry the organic layer.
e ltrates were collected and le to evaporate in a beaker
on a hot plate [].
e crude extract was prepared from the dried powder of
the leaves and owers using methanol as the extraction sol-
vent.Intheratioof:,gofthepowderedJ. sambac leaves
and owers was soaked in  mL methanol, respectively.
e mixture was then subjected to agitation using magnetic
stirrer for  hours at room temperature. e mixture was
then ltered using the Whatman lter paper No.  whereby
the ltrate obtained was collected. e process was repeated
using the remaining residue with  mL methanol. Both
ltrates were then mixed and concentrated under reduced
pressure using a rotary evaporator. e extracts obtained were
nally pounded to dryness under the fume hood in order to
produce a crude methanol extract [].
e essential oil and methanol extract were dissolved
with % DMSO whereas the aqueous extract was dis-
solved in sterile distilled water to nal concentration of
%, %, %, and % for essential oil and  mg/mL,
 mg/mL,  mg/mL,  mg/mL,  mg/mL,  mg/mL,
and  mg/mL for methanol extract. All the extracts were
sterilized by passing through a . 𝜇m pore membrane lter.
2.3.PreparationofMicroorganismStrain. e skin samples
were collected from the subjects under the approval of the
Faculty Research Committee Code NN--. Using a
sterile scalpel blade, the samples were placed in sterilized
plastic bags for storage and transport to the laboratory. e
fungal strains isolated from skin samples were mounted in
% KOH, glycerol, Parker ink solution, and lactophenol
cotton blue to identify the samples. It was also tested with
% H2O2to investigate the presence of catalase which
is an indicator of Malassezia sp. It was then inoculated
on Sabouraud dextrose agar (SDA) plates overlaid with
 mL olive oil as a source of lipid to which penicillin and
streptomycin combination (PenStrep) was added to inhibit
bacterial growth. e plates were incubated for  hours at
C aer which the colonies were examined and studied.
e colony was identied as Malassezia sp. aer undergoing
macroscopic colony test on SDA, microscopic observation by
Gram staining, and Tween assimilation test [].
2.4. Inoculum Preparation. e inoculum preparation of
yeast suspension was standardized using a spectrophotome-
ter. e optical density of the yeast suspension was adjusted to
turbidity at absorbance (A) reading within the range of .
at  nm which corresponded to 8CFU/mL whereas for
the lamentous fungi, the same inoculum size was equivalent
to absorbance (A) reading adjusted within the range of .
to . at  nm. Tween  was added as wetting agent to
facilitate the preparation of inoculum [].
2.5. Screening of Antimicrobial Activity. e extracts from the
owers and leaves of J. sambac were subjected to antifungal
screening by agar disc diusion method []. SDA plates
which were overlaid with mL of olive oil were inoculated
with the test isolate by spreading the standardized inoculum
on the surface of the agar plate with sterile swab. Holes of
diameter  mm were punched onto the sterile lter paper
and soaked with 𝜇Lofeachoftheextractsolutionsat
 mg/mL,  mg/mL,  mg/mL, and mg/mL. Flucona-
zole disc ( mg/mL) served as positive control whereas the
disc containing % DMSO alone was used as a negative
control for antifungal assay. e discs were dried for  hours
and placed onto the agar plates. All the plates were incubated
at C for  hours in moist condition because this is
the optimum growth temperature for all the fungi tested.
Journal of Mycology
e antimicrobial activity was assessed by measuring the
diameter of the zone of inhibition in mm from observation of
the clear zones formed surrounding each disc. e bioassay
was performed in triplicate in order to calculate the mean
value.
2.6. Determination of Minimum Inhibitory Concentration.
e methanol extract from the owers and leaves of J.
sambac was subjected to antifungal sensitivity testing by
broth microdilution method []. e -microtiter well was
prepared by dispensing  𝜇LofSDAbrothoverlaidwithmL
of olive oil and le for  minutes before adding  𝜇Lofthe
yeast suspension into each well. One hundred 𝜇Lfromthe
stock solution of J. sambac essential oil at % concentration
was added into the rst well, then followed by  𝜇Lof%,
%, and % essential oil added to the next three wells
consecutively.eplatewascoveredwithasterileplateshaker
at  rpm for  seconds and then incubated at Cfor
 hours. At the end of the incubation period, the plates
were evaluated for the presence or absence of growth. MIC
is the lowest concentration of the antifungal agent showing
no turbidity aer  hours, where the turbidity is interpreted
as visible growth of the fungi. e bioassay was performed in
triplicate in order to calculate the mean value.
3. Results
e skin samples obtained from the patients showed cell
morphology bearing the hyphae that resembled the shape
of spaghetti and meatball. e samples were tested positive
forcatalaseduetothepresenceofgasbubblesinreactionto
% hydrogen peroxide (H2O2).Outoftheveskinsamples,
 isolates were shown to be similar to the morphology of
Malassezia sp. e isolates were named M, M, and M,
respectively, with M and M growing on SDA with olive
oil and M without olive oil. e rst isolate (M) showed
a creamy white and smooth colony, while the second (M)
isolateshowedacreamypinkandsmoothcolony,bothon
SDAwitholiveoil.ethird(M)isolateshowedacreamy
white and smooth colony on SDA without olive oil. All
isolates were then observed microscopically by Gram staining
using a microscope. e morphology of cells according to
isolates was presented in Figures .Allisolatesshowed
budding in the cells with M (Figure )andM(Figure )
showingbroadbasedbuddingwhileM(Figure )showing
narrow based budding. Tween assimilation test indicated a
pos itive g row t h on Twee n  , Tween  , and Twe e n  for
M (Figure ) an d posit ive gr owth o n all the Twe en , Twe e n
,Tween,andTweenforMisolate(Figure ). M
isolate, however, showed negative growth for all the Tween
concentration as observed in Figure .
Table  showed the percentage yield of methanol extract
and essential oil from both the leaves and owers of the plant.
e extraction yield using methanol showed that the owers
of J. sambac produced .% crude extract whereas the yield
from leaves of J. sambac was only .%. On the other hand,
it was the leaves of the plant which produced a higher yield
of essential oil (.%) compared to its owers, whereby only
50 𝜇m
F : Cells from M isolate with broad based budding (Gram
staining, magnication x).
50 𝜇m
F : Cells from M isolate with broad based budding (Gram
staining, magnication x).
.% of the essential oil was obtained using hydrodistillation
technique.
Table  showedtheresultofantifungalactivityofthe
methanol extract from the leaves and owers of J. sambac.
All the isolates were susceptible towards the methanol extract
from both parts of the plant with mean inhibition zone
ranging from 9.10 ± 1.92mm to 12.90 ± 1.68 mm. M isolate
showed the highest susceptibility towards the methanol
extract from both the leaves and owers with inhibitory
zone of 11.10 ± 1.92mm and 12.90 ± 1.68 mm, whereas
the methanol extract from J. sambac leaves and owers
exhibited the lowest inhibitory zone of 9.10 ± 1.92mm and
10.17 ± 0.38mm, respectively against M isolate. Results of
MIC value determination using microdilution assay showed
that both the extracts from J. sambac have equal antifungal
ecacy towards the M and M isolates with MIC values
of the leaves extract and ower extract at, respectively,
 mg/mL and mg/mL. is indicated that methanol
extract of owers exhibited twice inhibitory eect against
Malassezia sp. compared to leaves. On the other hand, M
isolate displayed weaker activity with MIC value of methanol
extract from J. sambac leaves and owers of  mg/mL and
 mg/mL, respectively.
Table  showedtheresultofantifungalactivityofthe
essential oil from the leaves and owers of J. sambac. Disc
diusion screening as measured by zone of inhibition showed
that essential oil from J. sambac owers produced mean inhi-
bition zone ranging from 9.36±1.22 mm to 13.06± 0.26mm.
Journal of Mycology
50 𝜇m
F : Cells from M isolate with narrow based budding (Gram
staining, magnication x).
Tween 80
Tween 60
Tween 40
Tween 20
F : Tween assimilation of M isolate showing positive
reaction towards Tween , Tween , and Tween .
T : Percentage yield of methanol extract and essential oil from
the leaves and owers of J. sambac.
Parts of the plants Percentage yield (%)
Methanol extract Essential oil
Leaves . .
Flowers . .
No antifungal activity was observed from the essential oil
from the leaves of J. sambac as it did not produce any
inhibitory eect against all isolates tested. e MIC values of
essential oil from J. sambac ower against all isolates were in
arangeof%to%outof%in𝜇Lofessentialoil.
It was also clearly demonstrated that J. sambac owers have
the same antifungal ecacy towards both M and M isolates
with MIC value of % strength of the essential oil.
4. Discussion
Although Malassezia is an opportunistic fungus in normal
ora, it can be pathogenic and causes a variety of skin
disorders which utilizes the lipid produced among humans
due to the increasing population and global temperature
[]. Although these diseases were treatable with commercial
antifungals, the need to continually use them over a long
Tween 20 Tween 40
Tween 80
Tween 60
F : Tween assimilation of M isolate showing positive
reaction towards Tween , Tween , Tween , and Tween .
Tween 80
Tween 60
Tween 40
Tween 20
F : Tween assimilation of M isolate showing negative
reaction towards Tween , Tween , Tween , and Tween .
period of t ime results in many side eects and can be the cause
of increased resistance [,].
e search for antimicrobial from natural sources has
received much attention and eort has been put in to identify
compounds that can act as suitable antimicrobial to replace
synthetic agent. Natural plants have been used in many coun-
tries following remedies from tradition and custom due to
its abundance and availability. Phytochemicals derived from
plant product serve as a prototype to develop more eective
medicine in controlling the growth of microorganism with
less toxicity []. roughout the history of mankind, many
infectious diseases have been treated with plant extract. e
extraction of extract from medicinal plant has shown that
some of the screened plants are potentially rich source of
antibacterial and antifungal agent [,].
In the present study, the skin samples obtained from
vesubjectswereculturedtoisolateMalassezia sp. From
the ve samples, only three isolates of fungi were obtained
which showed resemblance to Malassezia sp. Microscopical
observation showed broad based budding of cells in M
and M isolates whereas M showed narrow based budding.
ebroadbasedbuddingofMandMisolatesmimicked
the morphology of cells represented by Malassezia sp.[].
Journal of Mycology
T : Antifungal activity of methanol extract from the leaves and
owers of J. sambac against M. sympodialis, M. dermatitis, or M.
furfur and non-Malassezia species.
Isolate Parts of the
plants
Zone of
inhibition
(mm ±SD)
MIC value
(mg/mL)
M
Leaves . ±. 
Flowers . ±. 
Positive
control . ±. 
M
Leaves . ±. 
Flowers . ±. 
Positive
control . ±. 
M
Leaves . ±. 
Flowers . ±. 
Positive
control . ±. 
PositivecontrolusedwasFluconazoleatmg/mL.ediameterof
inhibition zone was presented as a mean of  replicates. e isolates M, M,
and M represented M. sympodialis, M. dermatitis,or M. furfur and non-
Malassezia species, respectively.
T : Antifungal activity of essential oil from the leaves and
owers of J. sambac against M. sympodialis, M. dermatitis, or M.
furfur and non-Malassezia species.
Isolate Parts of the
plants
Zone of
inhibition
(mm ±SD)
MIC value
(%)
M
Leaves —
Flowers . ±. 
Positive
control . ±. 
M
Leaves —
Flowers . ±. 
Positive
control . ±. 
M
Leaves —
Flowers . ±. 
Positive
control . ±. 
Positive control used was Fluconazole at  mg/mL. e diameter of inhibi-
tion zone was presented as a mean of  replicates. — denotes no inhibition of
bacterial growth. e isolates M, M, and M represented M. sympodialis,
M. dermatitis,or M. furfur and non-Malassezia species, respectively.
All isolates were tested with Tween assimilation test which
acts as a lipid provider for the fungus. M isolate showed
positive growth towards Tween , Tween , and Tween
 whereas positive growth was recorded by M isolate in
all Tween. is indicated that M isolate could possibly be
M. sympodialis whileMisolateshowedthepossibilityof
either M. dermatitis or M. furfur species. M isolate showed
negative growth towards all Tween and is therefore possibly
M. globosa or a non-Malassezia species. However, since M.
globosa needed lipid for its growth and isolation and M is
isolated from SDA without olive oil, M can be conrmed as
anon-Malassezia species. From these observations, M and
M isolates were conrmed to be Malassezia sp. whereas M
was identied as a non-Malassezia species.
e result of extraction yield of the J. sambac showed
that methanol was capable of extracting the high quantity of
constituents from the owers of J. sambac whereasmostofthe
essential oil was produced by the leaves of the plant. e high
percentage yield of methanol extract indicated that methanol
was the best solvent that can extract many of the polar active
compounds found in the owers compared to the leaves of J.
sambac. e polarity of methanol and the solubility of plant
secondary metabolites in methanol could be the probable
reason for the high extractive value of methanol extract [].
Hydrodistillation however produced higher yield of essential
oil from the leaves of J. sambac compared to the owers. is
is supported by the nding that an essential oil is distilled
from the fermented and dried leaves of Camellia sinensis,a
source of the popular beverage tea [].
Results obtained from the screening of antifungal activity
indicated that the methanol extract from both the leaves
and owers of J. sambac showed inhibitory eect against all
isolates tested. However, Malassezia sp. represented by M
andMisolatesappearedtobemoresusceptibletowardsthe
eect of J. sambac owers which yield more phytocompo-
nents extracted by methanol. e methanol ower extract
showed higher yield and higher antifungal activity than the
methanol leaves extract. Hence, it can be noted that as for
methanol extraction the higher the extractive potential, the
stronger the antifungal activity. However this is not the case
for the hydrodistillation method because despite the high
yield of essential oil from J. sambac leaves compared to its
yield from the owers, the former showed no inhibitory eect
against all three isolates studied. is means that Malassezia
sp. represented by M and M isolates was only susceptible to
the essential oil from J. sambac owers which might contain a
promising antifungal phytoconstituent against Malassezia sp.
e inability of essential oil of leaves to exhibit any inhibitory
eect could mean that hydrodistillation method was unable
to extract the active antifungal compound eciently from
the leaves of the plant []. In other words, this method of
essential oil extraction is slightly ineective if used on other
parts of the plant other than the owers. is is supported
by []onmultipleplants,whichstatedthathydrodistillation
on other parts of a plant besides owers produces less
antimicrobial activity. However, there is no past research in
order to make comparison that reports the antimicrobial
potential of other parts of J. sambac besides owers and
therefore, this nding is interesting because it was shown that
essential oil from other parts of J. sambac besides owers
cannot be exploited as a source of antimicrobial agent.
e presence of antifungal activity in the methanol
extract from both the leaves and owers of Jasminum sambac
may be due to the presence of tannin. J. sambac owers
and leaves were reported to contain the polyphenol tannin
and sambacin which are known to possess antimicrobial
property []. However the essential oil from J. sambac
owers which comprise mainly 𝛼-farnesene, benzyl acetate,
Journal of Mycology
and linalool [] could contribute to its anti-Malassezia
activity.
5. Summary
is study demonstrated that both the methanol and essential
oil from the owers of Jasminum sambac showed promising
inhibitory eect against Malassezia sp. isolated from the
skin samples of patients with dandru. In conclusion, J.
sambac owers can be developed as potential phytother-
apeutic source of treatment against Malassezia-associated
skin infections and possibly as an additive ingredient in the
development of medicated shampoo against dandru as well
as skin and scalp infections.
6. Recommendation
However, further investigation to evaluate the antifungal
activity of J. sambac in mice infected with Malassezia sp. is
necessary to correlate the eectiveness of its methanol extract
and essential owers in vivo.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgment
is research was funded by the Ministry of Higher Educa-
tion, Government of Malaysia, under the research Grant code
FRGS///SG/UKM//.
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