Original Article
`
`Pharm Sci Asia 2017; 44 (4), 209-216
`DOI : 10.29090/psa.2017.04.209
`
`Efficacy of essential oil formulations against malodor causing
`bacteria
`P. Khuntayaporn1*, J. Suksiriworapong2,3
`1 Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand
`2 Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand
`3 Center of Excellence in Innovative Drug Delivery and Nanomedicine, Faculty of Pharmacy,
` Mahidol University, Bangkok, Thailand
`
`Abstract
`
`Malodor is an unpleasant sense induced by overgrowth of malodor causing bacteria. These
`bacteria can produce volatile organic compounds from their normal metabolisms. To reduce malodor,
`good hygiene combined with antimicrobial agents are suggested resulting in lowering amount of foot
`odor-producing bacteria. Essential oils have many favorable properties including unique senses and
`antimicrobial activities. In this study, eight essential oils were tested against five malodor causing
`bacteria. From the results, lemongrass oil exhibited the lowest MICs and MBCs. Meanwhile, clove
`oil and cinnamon leaf oil showed lower potency than lemongrass oil. Therefore, lemongrass oil was
`selected to be developed self-emulsifying formulations for foot bath. Four surfactants were chosen to
`test the compatibility with lemongrass oil. Only Tween 20 and Span 20 gave clear appearance and no
`phase separation after mixed with the oil and they were combined to be used in the formulation. The
`highest concentration of lemongrass oil in the formulation was 40% w/w. Tween 20 and Span 20 were
`mixed at various ratios ranged from 50:10% to 10:50% w/w. After mixing with water, all formulations
`could be simply emulsified with the particle size of less than 200 nm and no phase separation was
`observed. The formulation containing 40:50:10% w/w of oil:Tween 20:Span 20 demonstrated the
`most potent antibacterial activity and its MICs and MBCs were lower than lemongrass oil alone
`due to the formation of very fine emulsion. This formulation showed promising potential for the
`development of foot bath self-emulsifying emulsion formulation to be used in Thai spa.
`
`Keyword: Essential oil, antibacterial activity, self-emulsifying, malodor causing bacteria, foot bath
`
`1. INTRODUCTION
`
`The layers of skin are keeping moist
`in the body and protecting body from harmful
`environments including pathogens. Skin contains
`two types of sweat glands which are eccrine
`and apocrine glands. Eccrine glands are found
`all over the body especially on the palms and
`feet while apocrine glands can be found at
`specific areas such as armpits. Skin is also the
`living place for normal flora bacteria. These
`bacteria in beneficial surrounding by secretions
`from sweat glands create body odor. In particular
`to feet and armpits where high density of
`microorganisms are living, the bacteria produce
`unpleasant odor frequently resulting in
`uncomfortable sense of human. Malodor can be
`
`*Corresponding author: piyatip.khn@mahidol.edu
`
`caused by bacteria overgrowth in optimum
`environment1. To reduce odor, some strategies
`such as, good hygiene combined with deodorant,
`antiperspirant or even antimicrobial agents,
`were suggested to decrease amount of bacteria1.
`Some species of bacteria were reported to release
`specific characteristic smell in vitro caused by
`their normal metabolism.
`
`Volatile organic compounds (VOCs),
`including fatty acids and their derivatives,
`such as isovaleric acid and isobutyric acid
`were the primary components of foot odor2.
`Those fatty acids were produced by many
`microorganisms such as Staphylococcus
`epidermidis, Staphylococcus aureus, Bacillus
`spp.3. Normal bacteria which can be found on
`
`Petitioner Dr. Squatch
` Ex. 1021
`
`

`

`210
`
`human skin are S. aureus and Coryneform bacteria
`such as, Corynebacterium spp., Brevibacterium
`spp., and Propionibacterium acnes4, 5. Rennie
`PJ, et al., reported Corynebacterium spp. and
`Brevibacterium spp. involving body malodor
`while Micrococcus spp. and Propionibacteria
`involved foot odor production6. Marshall J, et al.,
`mentioned that Micrococci, Staphylococci and
`aerobic Coryneform bacteria with the ability
`to produce exoenzymes such as lipase and
`proteinase were associated with foot odor7.
`Among these skin normal flora bacteria,
`Staphylococcus spp. have been well studied8.
`However, there were only a few studies of
`essential oils against Micrococcus spp. and
`Brevibacterium spp..
`
`Essential oils are volatile substances
`and can be obtained from various parts of plants.
`Essential oils have many favorable properties
`such as their unique aroma, anti-inflammatory,
`antioxidant and antimicrobial activities, which
`make them popular in many industries including
`pharmaceutical, food, fragrance, and dermatology8, 9.
`Hydrophobicity of essential oils plays an
`important role in antimicrobial activity. They
`enable to partition to the lipid parts of bacterial
`cell membrane leading to the death of microbes10.
`Antimicrobial activity of essential oils varies
`depended on types of assays and microorganisms.
`
`Hydrophobic property of essential oils
`makes them incompatible with water. To prepare
`a formulation containing essential oils, surfactants
`are required. Although many surfactants can
`solubilize the essential oils, however they
`reportedly affect the activity of essential oils11, 12.
`Tween 80 was often used as an emulsifier in
`emulsion-based formulations of essential oils. Ma
`Q, et al., reported the decrease of antimicrobial
`effect of eugenol when mixed with Tween 8013.
`Different surfactants can also exhibit different
`antimicrobial activity of formulations. Hammer
`KA, et al., demonstrated different surfactants
`affected inhibitory activity of tea tree oil such
`as, Tween 80 increased MICs of tea tree oil
`more than Tween 2014. In addition, increasing
`of the concentration of surfactants including
`Tween 80 in the formulations also decreased
`antimicrobial activity13, 15. Therefore, the
`objectives of this study were to test the
`
`antibacterial activity of essential oils and to
`determine proper surfactants which could
`maintain antibacterial activity of essential oils.
`
`2. MATERIALS AND METHODS
`2.1. Materials
`
`All plant essential oils were purchased
`from Thai-China Flavours and Fragrances
`Industry Co., Ltd, Thailand. The oils employed
`in this study were lemongrass oil (Cymbopogon
`citratus (DC.) Stapf, part used: leaves), galanga
`oil (Alpinia galanga L., part used: rhizomes),
`kaffir lime oil (Citrus hystrix L., part used:
`leaves), holy basil oil (Ocimum tenuiflorum L.,
`part used: leaves), sweet basil oil (Ocimum
`basilicum L., part used: leaves), cinnamon oil
`(Cinnamomum zeylanicum, part used: leaves),
`clove oil (Syzygium aromaticum L., part used:
`buds) and turmeric oil (Curcuma longa L., part
`used: rhizomes).
`
`2.2. Bacterial culture collection
`
`Microorganisms used in this study were
`chosen based on skin normal flora. Bacillus
`subtilis ATCC6633, Micrococcus luteus
`ATCC9341, Staphylococcus epidermidis ATCC
`14990, Staphylococcus aureus ATCC25923,
`and Brevobacterium spp. were used in this
`study. Brevibacterium spp. was purchased from
`the culture collection of the Thailand Institute of
`Scientific and Technological Research (TISTR).
`Optimum culture temperature of all tested
`microorganisms was 37°C except for Brevibac-
`terium spp. which required at 30°C. All bacteria
`were cultured in tryptic soy broth. For the
`determination of minimum inhibitory concen-
`trations (MICs), Mueller Hinton broth was
`used as a culture medium.
`
`2.3. Antimicrobial activity assay of essential oils
`
`The stock solution of the essential
`oils were prepared at 1% v/v by dissolved in
`DMSO and then diluted with water. DMSO
`was used at the limit of not more than 5%v/v
`of total volume. The MICs were determined
`by broth microdilution assay. Briefly, single
`isolated colony was selected and cultured
`
`P. Khuntayaporn et al.
`
`

`

`overnight. Bacterial cultures were adjusted to
`0.5 McFarland and diluted to 106 CFU/ml. The
`stock solution of essential oils was diluted by
`two-fold dilution with Mueller Hinton broth
`and then the diluted bacterial culture was
`added. After 16-18 h of incubation, micro wells
`with clear solution were further determined for
`minimum bactericidal concentrations (MBCs)
`by streaking on Mueller Hinton agar. The agar
`plates were incubated overnight. The lowest
`concentration with no visible growth of bacteria
`was recorded as MBC.
`
`2.4. Determination of compatibility of
`surfactants and essential oils
`
`To formulate the small size emulsion of
`essential oils, the compatibility of surfactants
`and essential oils was determined. Span 20,
`PEG 400, Solutol HS15 and Tween 20 were
`chosen since they were often used in micro- and
`nanoemulsion formulations. Lemongrass oil
`was mixed with each surfactant at 1:9, 5:5 and
`9:1 mass ratios. The appearance of the mixture
`was observed with naked eyes. The surfactant
`that gave the clear solution of mixtures was
`further utilized for the formulation.
`
`2.5. Preparation of self-emulsifying emulsion
`formulation
`
`A pair of surfactants which gave the
`clear solution was used to prepare self-emulsifying
`emulsion formulation. Lemongrass oil was
`employed in the formulation at 40% w/w while
`the rest component was the mixture of Tween
`20 and Span 20 at different ratios varied from
`50:10 to 10:50% w/w. The obtained formulations
`were then tested upon dilution with water at
`1:160 volume ratio. The appearance of formula-
`tions was observed before and after dilution.
`In addition, phase separation and particle size
`of formulations after dilution were examined.
`The particle size of stable formulations was
`measured by Zetasizer NanoZS (Malvern
`Instrument, Malvern, UK) at an angle of 173°,
`25°C.
`
`2.6. Antimicrobial activity assay of formulation
`
`The stable formulations with maximum
`
`211
`
`ratio of each surfactant were selected to
`test for their antimicrobial activity by broth
`microdilution assay. The formulations were
`diluted with water at 1:160 volume ratio
`yielding the final concentration of lemongrass
`oil of 0.25% v/v. After that, the diluted
`formulations were employed in antimicrobial
`assay to determine MICs and MBCs as
`previously described. MICs and MBCs were
`calculated and reported as amount of essential
`oil in % v/v.
`
`2.7. Statistical analysis
`
`All experiments were performed in
`triplicate. Descriptive statistics was performed
`in this study using Microsoft Excel 2010. All
`results are given as mean ± standard deviation.
`
`3. RESULTS AND DISCUSSIONS
`3.1. Antimicrobial activity assay of essential oils
`
`MICs and MBCs of tested essential oils
`against malodor causing bacteria were shown
`in Tables 1 and 2. Lemongrass oil demonstrated
`the lowest MICs against all tested microorganisms.
`MICs of lemongrass oil ranged from 0.0312%
`v/v to 0.125% v/v. The MICs of clove oil and
`cinnamon leaf oil were ranked as the second
`and third lowest MICs values against all tested
`bacteria and ranged from 0.125% v/v to 0.25%
`v/v and 0.125% v/v to 0.5% v/v, respectively.
`Sweet basil oil exhibited inhibitory effect
`against B. subtilis and Brevibacterium spp. at
`0.5% v/v whereas turmeric oil and kaffir lime
`oil showed the effect against only B. subtilis
`and S. epidermidis, respectively, at the same
`concentration. However, galangal oil and holy
`basil oil at the maximum tested concentration
`did not demonstrate inhibitory activity against
`all tested bacteria. All essential oils exhibited
`antimicrobial activity were further studied for
`MBCs. The MBCs of lemongrass oil were
`found to be in the range of 0.0625-0.25% v/v
`depended on the species of bacteria (Table 2).
`Nevertheless, this oil did not exhibit bactericidal
`activity against S. epidermidis at the maximum
`tested concentration of 0.5% v/v. Clove oil and
`cinnamon leaf oil demonstrated bactericidal
`activity against B. subtilis at 0.125% v/v and
`
`Efficacy of essential oil formulations against malodor causing bacteria
`
`

`

`212
`
`0.5% v/v, respectively. Meanwhile MBCs of
`these oils against other microorganisms were
`higher than 0.5% v/v. Sweet basil oil showed
`
`MBC against Brevibacterium spp. at 0.5% v/v
`while turmeric oil and kaffir lime oil showed
`higher than 0.5% v/v against the tested bacteria.
`
`Table 1. Minimum inhibitory concentration (%v/v) of essential oils against tested microorganisms
`
`
`
`
`
` Lemongrass oil
` Clove oil
` Cinnamon leaf oil
` Sweet basil oil
` Tumeric oil
` Kaffir lime oil
` Galanga oil
` Holy basil oil
`
`Minimum inhibitory concentration (%v/v)
`B. subtilis Brevibacterium spp. M. luteus S. epidermidis S. aureus
`0.0312%
`0.125%
`0.0625%
`0.0625%
`0.125%
`0.125%
`0.125%
`0.25%
`0.25%
`0.25%
`0.5%
`0.125%
`0.5%
`0.5%
`0.5%
`0.5%
`0.5%
`>0.5%
`>0.5%
`>0.5%
`0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
`
`Table 2. Minimum bactericidal concentration (%v/v) of essential oils against tested microorganisms
`
`Minimum bactericidal concentration (%v/v)
`
`
`B. subtilis Brevibacterium spp. M. luteus S. epidermidis S. aureus
`
`
`0.0625%
`0.25%
`0.0625%
`>0.5%
`0.125%
` Lemongrass oil
`0.125%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
` Clove oil
`0.5%
`>0.5%
`>0.5%
`>0.5%
`>0.5%
` Cinnamon leaf oil
`ND
`0.5%
`ND
`ND
`ND
` Sweet basil oil
`>0.5%
`ND
`ND
`ND
`ND
` Tumeric oil
`ND
`ND
`ND
`>0.5%
`ND
` Kaffir lime oil
`ND
`ND
`ND
`ND
`ND
` Galanga oil
`ND
`ND
`ND
`ND
`ND
` Holy basil oil
`ND; not determined due to MICs >0.5% v/v
`
`Brevibacterium spp. and Micrococcus
`
`spp. are bacteria living on the skin and they can
`cause a unique smell. However, there were a few
`studies of essential oils and very rare report on
`Thai essential oils against these bacteria. Van
`Vuuren, SF, et al., reported MICs of kanuka oil
`(Kunzea ericoides) and manuka oil (Leptospermum
`scoparium) against Brevibacterium spp. which
`were equal or less than 1 mg/ml16. In this study,
`MICs of essential oils against Brevibacterium
`spp. and M. luteus were determined. Lemongrass
`oil demonstrated the most potent agent against
`
`these bacteria. Orchard A, et al., also mentioned
`that lemongrass oil was one of promising
`essential oils against S. aureus8. Naik MI, et al.,
`also demonstrated MICs of lemongrass oil against
`some pathogenic bacteria included Bacillus spp.
`and S. aureus which were about 0.03 to 0.06%
`v/v17. From our results, lemongrass oil possessed
`the highest bacteriostatic and bactericidal
`activities against all tested bacteria. Clove oil
`and cinnamon leaf oil were ranked as the second
`and third effective agents, respectively. Hence,
`lemongrass oil was selected for further study.
`
`P. Khuntayaporn et al.
`
`

`

`213
`
`3.2. Compatibility study of essential oils and
`surfactants
`
`To prepare self-emulsifying formulation,
`the suitable surfactant is a key of success for
`the formulation development. According to the
`antimicrobial assay of essential oils, lemongrass
`oil was used in this study. Four surfactants
`were selected to study the compatibility with
`lemongrass oil. After mixing each surfactant,
`the transparency of mixture was evaluated by
`observing the letter through the mixture as
`
`illustrated in Figure 1 and then scored as ++, +
`or – indicating very clear, slightly clear and
`turbid, respectively. The results showed that no
`phase separation was observed for all lemon-
`grasss oil/surfactant mixtures. As summarized
`in Table 3, Tween 20 and Span 20 gave the
`clear mixture at all ratios. The mixture ratio
`at 5:5 showed the clearest solution. Therefore,
`Tween 20 and Span 20 were chosen for the
`formulation development at the lemongrass
`oil/surfactant ratio of 5:5.
`
`Figure 1. Naked eyes observation of clarity/turbidity of lemongrass oil/surfactant mixture.
`
`Table 3. Transparency results of the mixture of lemongrass oil and various surfactants
`
`
`
`
`
`
`
`Lemongrass
`oil:surfactant ratio
`1:9
`5:5
`9:1
`
`Tween 20
`
`Solutol HS 15
`
`PEG 400
`
`Span 20
`
`+
`++
`+
`
`-
`+
`++
`
`-
`-
`++
`
`+
`++
`+
`
`++, + and – denote very clear, slightly clear and turbid appearance.
`
`3.3. Self-emulsifying emulsion formulation
`
`Self-emulsying emulsion is a system
`that contains oil and surfactant/co-surfactant.
`When gently mixing with water, fine emulsions
`will be spontaneously formed14. Self-emulsifying
`emulsions are normally formed with a droplet size
`between 100-300 nm whereas self-emulsifying
`microemulsions are produced with transparent
`
`characteristics with a droplet size of less than
`50 nm14, 18, 19. From the compatibility results,
`two formulations consisting of lemongrass oil
`and Tween 20 or Span 20 were diluted with
`water by 160 folds on the basis of the purpose
`of foot bath formulation. However, the formed
`emulsions were unstable with subsequent
`phase separation (data not shown). In general,
`
`Efficacy of essential oil formulations against malodor causing bacteria
`
`

`

`214
`
`the fine self-emulsifying emulsions require both
`surfactant(s) and co-surfactant(s)18. Tween 20
`and Span 20 are typically used in combination
`for emulsion formulation. Therefore, Tween 20
`and Span 20 were combined in the formulation.
`All preparations containing lemongrass oil
`higher than 40% w/w became phase separation
`after mixed with water. Hence, the maximum
`concentration of lemongrass oil in the formu-
`lations was 40% w/w. The surfactant ratios
`of Tween 20 and Span 20 in the formulation
`
`were varied from 50:10 to 10:50% w/w. All
`formulations were able to emulsify with water
`and their appearance was slightly turbid. The
`particle size results are summarized in Table 4.
`All self-emulsifying emulsions possessed very
`small size with the particle size of less than
`200 nm. However, only the formulations con-
`taining 40:50:10 and 40:10:50% w/w of lem-
`ongrass oil:Tween 20:Span 20 were selected
`to further determine antimicrobial activity of
`the formulations.
`
`Table 4. Particle size of the formulations after mixing with 25°C water
`
`
`
`
`
`
`
`%Weight of oil:Tween 20:Span 20
`in the formulations
`40:10:50
`40:30:30
`40:50:10
`
`3.4. Antimicrobial activity assay of the for-
`mulation
`
`The 40:50:10 and 40:10:50% w/w
`lemongrass oil:Tween 20: Span 20 formulations
`were mixed with water in 1:160 ratio prior
`to determine antimicrobial activity by broth
`microdilution assay. MICs of both formulations
`are shown in Table 5. From the results, MICs
`against all tested bacteria of the 40:50:10%
`w/w formulation was lower than those of the
`40:10:50% w/w formulation. This was attributed
`to the change in particle size of the formulation
`after mixing with warm water (40 °C) compared
`to ambient water as a result of the solubility
`change of surfactants in warm water. The
`formulation with lower amount of Tween 20
`had 10-times increased particle size (1116±658
`nm) after mixing with warm water. Meanwhile
`the higher amount of Tween 20 maintained the
`particle size under 200 nm (187±28 nm). The
`lemongrass oil in the 40:50:10% w/w formula-
`tion was more potent than intact lemongrass
`oil against almost all tested bacteria while that
`in 40:10:50% w/w formulation showed little
`potency. However, MIC of S. epidermidis in
`40:50:10% w/w formulation slightly increased
`from that of lemongrass oil. It has been reported
`
`Particle size (nm)
`
`141±1
`146±1
`148±2
`
`that micro- or nanoemulsions could enhance
`antibacterial activity of essential oils as a
`consequence of very small droplet size which
`could increase stability, surface area and
`biological activity of essential oils19. Therefore,
`the enhanced antimicrobial activity of 40:50:10%
`w/w formulation was attributed to the formation
`of very small size of emulsion. This formulation
`showed promising potential to develop as foot
`bath formulation to reduce foot odor-producing
`bacteria.
`
`4. CONCLUSION
`
`Malodor is an unpleasant sense which
`caused by normal skin bacteria combined
`with their optimum environment. Essential
`oils consisting of natural volatile substances
`possess unique sense and several biological
`activities including antimicrobial activity. In
`this study, lemongrass oil exhibited the most
`potent bacteriostatic and bactericidal activity
`against foot odor-causing bacteria with the
`lowest MICs and MBCs. When formulated in
`self-emulsifying emulsion for foot bath, Tween
`20 and Span 20 illustrated good property for
`this purpose. The combination of Tween 20
`
`P. Khuntayaporn et al.
`
`

`

`215
`
`and Span 20 was used in the formulation since
`they produced the stable and small size emulsion
`after mixing with water at 1:160 volume ratio.
`The formulation containing 40:50:10% w/w of
`lemongrass oil:Tween 20:Span 20 exhibited
`small particle size of less than 200 nm, stable
`
`emulsion after mixing with both ambient and
`warm water and more potent antimicrobial
`activity as compared to lemongrass oil alone.
`However, further improvement on formulation,
`stability and antimicrobial activity is needed
`and is being under our investigation.
`
`Table 5. MICs and MBCs (% v/v) of lemongrass oil in different formulations against tested microorganisms
`
`Ratio
`
` (Oil:tween 20:Span 20)
`
`40 : 10 : 50
`
`
`
`
`
`
`
`
`
`40 : 50 : 10
`
`
`
`
`
`
`
`
`
`
`Microorganisms
`
`
`B. subtilis
`Brevibacterium spp.
`M. luteus
`S. epidermidis
`S. aureus
`B. subtilis
`Brevibacterium spp.
`M. luteus
`S. epidermidis
`S. aureus
`
`ND; not determined due to MIC >0.125% v/v
`
`Conflicts of interest
`
`The authors declare that there are no
`conflicts of interest.
`
`5. ACKNOWLEDGEMENTS
`
`This study was partially financial
`supported by Faculty of Pharmacy, Mahidol
`University, Bangkok, Thailand. Authors wish
`to thank Assoc. Prof. Mullika Chomnawang,
`Miss Cholthicha Waitayawijit, Miss Sumana
`Sincharoen and staffs in Department of micro-
`biology and Department of Pharmacy, Faculty
`of Pharmacy, Mahidol University for their
`assistance.
`
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