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Inhibitory effect of formulated lemongrass shampoo on malassezia furfur: A

yeast associated with dandruff

Article  in  The Southeast Asian journal of tropical medicine and public health · March 2011
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 EffEct of LEmongrass shampoo on Malassezia furfur

INHIBITORY EFFECT OF FORMULATED LEMONGRASS

SHAMPOO ON MALASSEZIA FURFUR: A YEAST
ASSOCIATED WITH DANDRUFF
Mansuang Wuthi-udomlert1, Ployphand Chotipatoomwan2, Sasikan Panyadee2
and Wandee Gritsanapan2

1
Department of Microbiology; 2Department of Pharmacognosy, Faculty of Pharmacy,
Mahidol University, Bangkok, Thailand

Abstract. Lemongrass (Cymbopogon citratus Stapf) has been used in cooking and in
many traditional medicines; the essential oil contains citral as a major constituent.
This study evaluated the antifungal activity of lemongrass oil against Malassezia
furfur, an opportunistic yeast associated with dandruff, by using a broth dilution
assay. From the minimum fungicidal concentration (MFC) obtained, the oil was
then incorporated at different percentages into shampoo formulations. The formu-
lated shampoos were kept at room temperature (28º-30ºC) and under accelerated
condition (45ºC). At the end of the irst and sixth weeks, after preparation, all
formulations were tested again and the appearance was recorded. Selection of an
appropriate formula was based on antifungal activity against M. furfur, the physi-
cal appearance, the chemical properties and stability of the formula. Two percent
lemongrass oil shampoo provided the required qualities necessary for commercial
use. After being kept for 6 weeks at 28º-30ºC and 45ºC, this formulated shampoo
gave MFCs against M. furfur of 75 µl/ml and 18.75 µl/ml, respectively.
Keywords: Cymbopogon citratus, dandruff, herbal shampoo, lemongrass, Malas-
sezia furfur

INTRODUCTION of Malassezia, M. globosa and M. restricta,

have also been reported in human dan-
Malassezia furfur is a common sap- druff (Gupta et al, 2004). Dandruff is a su-
rophytic, lipophilic yeast found on seba- pericial disorder of the stratum corneum
ceous areas of human skin: face, scalp, of the scalp, with cell hyper-proliferation
and upper trunk. This unicellular fungus cause laking, itching and redness. The
is associated with several skin disorders clinical appearance and symptoms are
that predominantly involve the supericial similar to seborrheic dermatitis, only the
layers of skin and exacerbates many der- latter is more severe. The symptoms de-
matologic diseases: tinea versicolor, seb- velop when the lipophilic yeast Malassezia
orrheic dermatitis, folliculitis and atopic takes up free fatty acids (FFAs) from se-
dermatitis (Levin, 2009). Other species baceous triglycerides. The FFAs penetrate
Correspondence: Wandee Gritsanapan, Depart- the stratum corneum breach the scalp skin
ment of Pharmacognosy, Faculty of Pharmacy, barrier and lead to transepidermal water
Mahidol University, Bangkok10400, Thailand. loss attributed to dandruff (Dawson et al,
Tel/Fax: 66 (0) 2644 8701 2005). The treatment of dandruff includes
E-mail: [email protected] application of topical antifungal or other

Vol 42 No. 2 March 2011 363

 southEast asian J trop mEd pubLic hEaLth

products. Since recurrences occur com- available scientiic evidence into daily use
monly, prophylaxis using products for products has been reasonably encouraged.
skin and hair to maintain good healthy This study focused on determining
skin and a healthy appearance is needed. the effective ingredient of the herb and
Lemongrass (Cymbopogon citratus the appropriate procedure to include that
Stapf), from the family Gramineae, is active ingredient into a shampoo against
an important Asian culinary herb that Malassezia furfur.
has been included in a wide range of
herbal products, household items and MATERIALS AND METHODS
traditional medicines. The principal con-
Plant materials
stituent of lemongrass essential oil is citral
(3,7-dimethyl-2,6-octadienal), a mixture of Lemongrass leaf sheaths were pur-
isomeric acyclic monoterpene aldehydes: chased from a market in Bangkok, Thai-
geranial (trans-citral, citral A) and neural land, in June 2008. Identiication of lem-
(cis-citral, citral B) with a small quantity ongrass was made by Prof W Gritsanapan,
of geraniol, geranylacetate and monoter- Department of Pharmacognosy, Faculty of
pene oleins (Simonsen and Owen, 1953; Pharmacy, Mahidol University. Specimens
Katsukawa et al, 2010). These constituents were kept at the Department of Pharma-
comprise 75-85% of lemongrass oil and are cognosy, Faculty of Pharmacy, Mahidol
a pale yellow liquid with a strong lemon- University.
like odor (FAO, 1967; Formacek and Ku- Volatile oil hydrodistillation
beczka, 1982). Because of these properties, Fresh lemongrass leaf sheaths were
citral is widely used as an essential raw cut into small pieces, put in a round bot-
material in pharmaceuticals, perfumery tom lask and distilled water was added
and cosmetics industries. Many studies (1:10). This was connected to a hydrodis-
have evaluated its biological properties, tillation instrument. The mixture of oil
such as antifungal activity against plant and water obtained was separated by a
and human pathogens (Yousef et al, 1978; separating funnel; the collected volatile
Asthana et al, 1992; Shadab et al, 1992; oil was kept in a tightly closed container
Rodov et al, 1995; Adegoke and Odesola, with light protection until used.
1996; Schwiertz et al, 2006; Tzortzaki and Lemongrass oil and citral
Costas, 2007; Tyagi and Malik, 2010a),
Zero point one milliliter each of lem-
bactericidal effects (Onawunmi et al, 1984;
ongrass oil and citral standard were mixed
Cimanga et al, 2002; Wannissorn et al, 2005;
in 1 ml of hexane, and spotted onto a thin
Lertsatitthanakorn et al, 2006; Schwiertz
layer chromatography aluminium sheet
et al, 2006; Naik et al, 2010; Tyagi and Malik,
of siliga gel GF254. The mobile phase was
2010a) and insecticidal properties (Rice
toluene:ethyl acetate 97:3. The developed
and Coats, 1994; Saddiq and Khayyat,
plate was observed under UV 254 nm and
2010). The WHO Model Formulary for
sprayed with vanillin sulfuric acid reagent
2008 (Stuart et al, 2009) lists it as a topical
(5% sulfuric acid in ethanol and 1% vanil-
dermatological medicine used in many
lin in ethanol).
proprietary shampoos with antifungal and
keratolytic properties against seborrheic Anti-malassezia activity
dermatitis. Thus, the integration of herb- Quantitative evaluation: broth dilution as-
als or traditional medicinal plants with say. The principle of the test was modiied

364 Vol 42 No. 2 March 2011

 EffEct of LEmongrass shampoo on Malassezia furfur

from reference method M27-A3 (CLSI,

2008) of the Clinical and Laboratory Stan-
dards Institutes (CLSI). An inoculum of
M. furfur was prepared from a mature
pure culture on Sabouraud dextrose agar
(SDA; Pronadisa, Madrid, Spain) using
Sabouraud dextrose broth (SDB; Pronadisa,
Madrid, Spain) to give 3x103-104 cfu/ml. A
twofold serial dilution of oil/shampoo was
made using SDB and dimethyl sulfoxide
(DMSO). A serial dilution of DMSO at simi-
lar concentrations was used as control. A
culture control was also included. All were a b a b
incubated at 28º-30ºC. The end point of the I II
test was recorded at the same time as the
culture control became positive. Subculture Fig 1–Comparison of thin layer chromato-
test concentrations yielded the minimum graphy of citral reference standard and
lemongrass oil employed in test sample.
inhibitory concentration (MIC) and the
I, TLC plate under UV 254 nm; II, TLC
minimum fungicidal concentration (MFC).
plate sprayed with vanillin-sulfuric acid
Formulation of shampoo containing lem- reagent; a, Citral reference standard; b,
ongrass oil Lemongrass oil sample.
Lemongrass oil was added to sham-
poo formulations at different concentra- activity by these formula was done after
tions. Other ingredients added to improve the irst and sixth weeks.
the quality and give the most appropri- Eficiency of formulated shampoo against
ate characteristics to the shampoo were M. furfur
Lolane complex (LC), EDTA, DI water, The accepted stable formulations
Texapon N 28, Dehyton K, sodium lauryl were tested for anti-malassezia activity
sarcosinate, comperlan KD and Glydant by broth dilution assay to test the ef-
plus. Twenty-ive percent citric acid and fectiveness the shampoos left at normal
sodium chloride were used to adjust the room temperature (28º-30ºC) and at high
pH and viscosity, respectively. temperature (45ºC).
To develop an anti-dandruff sham-
poo formula, lemongrass oil was added RESULTS
to different shampoo bases designated
MU001 and MU002 at 1, 2, 3, 4 and 5% Lemongrass oil and citral standard
w/w. These formulations were left at room The lemongrass oil used in the sham-
temperature for 1 and 3 weeks and the poo formulations was evaluated for its
formulas with an acceptable appearance active ingredients using thin layer chro-
were modiied into MU003 and MU004 at matography (TLC) at an Rf value of 0.38,
the same concentrations of lemongrass oil similar to the citral reference standard.
and kept at room temperature (28º-30ºC) A purple band appeared after spraying
or high temperature (45ºC) for 6 weeks. with vanillin-sulfuric acid, indicating the
Further evaluation of anti-malassezia presence of terpenes (Fig 1).

Vol 42 No. 2 March 2011 365

 southEast asian J trop mEd pubLic hEaLth

Table 1 Anti-malassezia activity of lemongrass oil

Anti-malassezia activities of lemongrass and citral standard
oil extract and citral standard against The activity of lemongrass oil and cit-
M. furfur. ral standard on M. furfur was investigated
Anti-malassezia by broth dilution assay. The inhibitory
activity (µg/ml) and fungicidal effects of lemongrass oil
Test sample
occurred at the same concentration (6.25
MIC MFC
µg/ml), half way between the inhibitory
Lemongrass oil extract 6.25 6.25 and fungicidal concentrations of the citral
Citral standard 3.13 12.50 standard (Table 1).

Table 2
Formulations of lemongrass oil shampoo.

Storage condition Qualityb

Formulationa
Weekc Temp Thickness Color Separation into MFCs
layers (µl/ml)

d
MU001 0 28º-30ºC ac ac ac
1 28º-30ºC ac ± ± -
MU002 0 28º-30ºC ± ac ac -
1 28º-30ºC ac ac un -
MU003 0 28º-30ºC ac ac ac 37.50-300.00
6 28º-30ºC ac ac ac 18.75-75.00
45ºC ac ac ac 9.38-37.50
a
Formulations MU003-B, MU003-X, MU004 and MU004-X were not included in the table.
b
Satisied result, ac, acceptable; un, unacceptable; ±, partially acceptable
c
0: refers to a freshly prepared sample
d
Not done

Table 3
MFCs of different percentages of lemongrass oil shampoo MU003 against M. furfur.

MFC (µl/ml) of shampoo at different

percentages of lemongrass oil
Duration in Temp
week(s) 1% 2% 3% 4% 5%

0 28º-30ºC 300.0 75.0 75.0 37.5 37.5

6 28º-30ºC 75.0 75.0 37.5 18.8 18.8
45ºC 37.5 18.8 18.8 18.8 9.4

MFC, minimum fungicidal concentration

366 Vol 42 No. 2 March 2011

 EffEct of LEmongrass shampoo on Malassezia furfur

Development of shampoo containing used in this study were similar to the citral
lemongrass oil. After the preliminary reference standard using its Rf value and
shampoo containing lemongrass oil was the similar color of the spots after spray-
formulated, it was further developed by ing with vanillin-sulfuric acid reagent,
adding or taking out of some compo- which revealed the presence of terpenes.
nents. The shampoo bases MU001 and Further studies of the chemical stability
MU002 consisted of different percentages of the formulae by quantitative analysis
of surfactant (C. Betaines), foam booster of active ingredients using HPLC/TLC
and thickener [cocamide MEA, hydroxy densitometry should be carried out.
ethylcellulose (HEC), NaCl] and various In order to develop a shampoo formu-
concentrations of lemongrass oil (1, 2, 3, la, the selection was based on the physical
4 and 5% w/w). After being left at room stability under room temperature and
temperature for one week, an acceptable high temperature (45ºC) to relect 2 years
combination of MU001 was modified of storage. Other properties included clear
by adding HEC to the shampoo base of solution, moderate thickness, and accept-
MU003 and MU004 and stored at 28º- able odor. In addition, lemongrass oil
30ºC and 45ºC for 6 weeks. Subformulae containing citral is also used as a natural
MU003-B, MU003-X, MU004, MU004-X bio-active component.
were also stored likewise before testing
The 2% lemongrass oil shampoo
for anti-malassezia activity.
formula MU003 was the most appropri-
The MU003 formula with 2% lemon- ate formula with required properties and
grass oil demonstrated the best stability anti-malassezia activity. This shampoo
under test conditions with good physical formula did not deteriorate over time and
appearance: clear solution, no separated exhibited anti-malassezia activity. The
layer, moderate thickness and acceptable MFC after one week at room temperature
odor (Table 2). Anti-malassezia activities was 75 µg/ml and at 45ºC was 18.75 µg/ml.
were determined under test conditions. This may be a result of losing water in the
After 6 weeks, MU003 formula with formula. The 2% concentration of lemon-
lemongrass oil demonstrated increasing grass oil was selected because the smell,
antifungal activity with increasing per- consistency and stability of the shampoo
centages of lemongrass oil (Table 3). were better than the others.

DISCUSSION ACKNOWLEDGEMENTS

Medicinal plants and crude herbal We would like to thank The Industrial
drugs have been included in traditional and Research Projects for Undergraduate
medicines and household remedies for Students (IRPUS) for their support and
a long time. Not all herbal preparations SC Artistry Co Ltd for advice on develop-
have been scientiically tested. In an at- ment of shampoo for formular.
tempt to determine the beneits of various
herbal extracts, we evaluated the effect of REFERENCES
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