PLoS One. 2015; 10(9): e0138602.
Published online 2015 Sep 25. doi: 10.1371/journal.pone.0138602
PMCID: PMC4583440
Christophe Egles, Editor
Abstract
Background
Calophyllum inophyllum
L. (Calophyllaceae) is an evergreen tree ethno-medically used along the
seashores and islands of the Indian and Pacific Oceans, especially in
Polynesia. Oil extracted from the seeds is traditionally used topically
to treat a wide range of skin injuries from burn, scar and infected
wounds to skin diseases such as dermatosis, urticaria and eczema.
However, very few scientific studies reported and quantified the
therapeutic properties of Calophyllum inophyllum oil (CIO). In
this work, five CIO from Indonesia (CIO1), Tahiti (CIO2, 3), Fiji
islands (CIO4) and New Caledonia (CIO5) were studied and their
cytotoxic, wound healing, and antibacterial properties were presented in
order to provide a scientific support to their traditional use and
verify their safety.
Methods
The
safety of the five CIO was ascertained using the Alamar blue assay on
human keratinocyte cells. CIO wound healing properties were determined
using the scratch test assay on human keratinocyte cells. CIO-stimulated
antibacterial innate immune response was evaluated using ELISA by
measuring β defensin-2 release in human derivative macrophage cells. CIO
antibacterial activity was tested using oilogramme against twenty
aerobic Gram- bacteria species, twenty aerobic Gram+ bacteria species,
including a multi-drug resistant Staphylococcus aureus strain and two anaerobic Gram+ bacteria species e.g. Propionibacterium acnes and Propionibacterium granulosum. To detect polarity profile of the components responsible of the antibacterial activity, we performed bioautography against a Staphylococcus aureus strain.
Results
Based
on Alamar Blue assay, we showed that CIO can be safely used on
keratinocyte cells between 2.7% and 11.2% depending on CIO origin.
Concerning the healing activity, all the CIO tested accelerated in vitro
wound closure, the healing factor being 1.3 to 2.1 higher compared to
control when keratinocytes were incubated after scratch with CIO at
0.1%. Furthermore, our results showed that CIO exhibit two distinct
antibacterial effects: one against Gram+ bacteria by direct inhibition
of mitotic growth and another potent effect against Gram- bacteria due
to increased release of β-defensin 2 peptide by macrophages.
Interestingly, the needed concentrations of CIO to inhibit bacteria
growth and to promote wound healing are lower than concentrations
exhibiting cytotoxic effects on keratinocyte cells. Finally, we
performed bioautography assay against Staphylococcus aureus to
determine polarity profile of the components responsible for CIO
antibacterial activity. Our results showed for the five tested CIO that
components responsible of the bacterial growth inhibition are the more
polar one on the TLC chromatographic profile and are contained in the
resinous fraction of the oil.
Conclusions
This
study was conducted to evaluate cytotoxicity, wound healing and
antibacterial properties of five CIO traditionally used to treat
infected wounds. Using cell and bacteria cultures, we confirmed the
pharmacological effects of CIO as wound healing and antimicrobial agent.
Moreover, we showed that concentration of CIO needed to exhibit
therapeutic effects are lower than concentrations exhibiting cytotoxic
effects in vitro. For the first time, this study provides
support for traditional uses of CIO. These wound healing and antibiotic
properties make CIO a valuable candidate to treat infected wounds
especially in tropical areas.
Introduction
Chronic
wounds remain a major health problem, particularly in tropical areas
where high temperature and humidity promote wound bacterial infections.
Increase in bacterial resistance to available antibiotics and poor
access to treatments worsen this issue [1].
Efforts are needed to find new antibiotics, easy to produce locally and
coming from cheap and renewable sources. Since years, plants have been
used in traditional medicine to treat a large range of human diseases [2,3].
Leaves and barks are generally prepared in infusion for internal use
and oil extracted from the fruits is rather used topically [4].
Among the natural products extracted from the plants that have
demonstrated biological activities, some of them need to receive
particular attention as antimicrobial and wound healing agents [5,6].
Indeed, new treatments accelerating wound closure and at the same time
preventing infections present a great interest, particularly in tropical
area. Calophyllum inophyllum is a medium to large evergreen tree presenting elliptical leaves, fragrant white flowers and large round nuts [7,8]. The tree is widely distributed along the coasts of the Indian and Pacific Oceans especially in Melanesia and Polynesia [9,10]. Locally called Tamanu in Polynesia, Tamanou de bord de mer in New Caledonia, Dilo in Fiji, Nyamplung in Indonesia this latter is traditionally used notably in the treatment of suppurating wounds [11–15]. The decoction of the leaves is employed to relieve dermatosis, urticaria and eczema [16], the juice of the bark is purgative [10], the first cold pressed seed oil is used in wound healing [13] and to relieve neuropathy associated with leprosy [13,17].
Oil is recommended for all kinds of burns, post-surgical and
suppurating wounds, dermatoses, acne, psoriasis, herpes, rheumatism and
gonorrhea [10,12,18,19]. Recently, a survey performed in the Marquesas archipelago reported that a preparation based on CIO named pani temanu is used to cure skin diseases related to itches, skin allergy, burns and mild wounds [20]. Despite this high therapeutic potential for skin disease, only few studies reported Calophyllum inophyllum oil (CIO) pharmacological properties. Pocidalo et al. described healing properties of CIO resin on animal experimental burns [21] and Patil et al. reported that compounds isolated from CIO possessed strong activity against HIV-1 [22]. In another one, four coumarin derivatives obtained from a crude extract of the nuts were shown to be active against Staphylococcus aureus [23]. More recently, Said et al. showed that CIO presents both UV-absorption and antioxidant properties [24].
Taken together these wound healing and antibiotic properties make CIO a
valuable candidate as an alternative therapeutic strategy to treat
infected wounds especially in tropical areas. In this study, five CIO
from Indonesia, Tahiti, Fiji islands and New Caledonia were compared for
their wound healing and antibacterial activities. Using whole oil
extracts on human cells and bacterial cultures, we attempt to ascertain
the safety of the studied CIO and to confirm their therapeutic effects.
Materials and Methods
Plant materials
Five
CIO from Indonesia (CIO1), Tahiti (CIO2, 3), Fiji islands (CIO4) and
New Caledonia (CIO5) were compared for their pharmacological properties.
The references, geographic origins and characteristics of studied oils
are summarized in supplementary material (S1 and S2 Tables). Calophyllum inophyllum
is not listed as endangered or protected species. In Indonesia, Tahiti
and Fiji islands, CIO is commercially exploited and no specific
permissions are required for scientific investigation. However, a
scientific research authorization is required for exploitation of New
Caledonia biological resources. In New Caledonia, nuts were collected
under the scientific research authorizations N°2188–2010 granted by
Department of Environment of the South Province. Oil production was
obtained following the manufacturer's guidelines, briefly: nuts were
collected after reaching maturity and fresh almonds were extracted from
the nuts. Then, fresh almonds were air dried before performing first
cold press before being filtrated and packaged under controlled
atmosphere. Refined Olive oil (Sigma-Aldrich) was used as control for
all the subsequent experiments.
CIO fatty acid analysis
Lipids
from CIO were extracted with chloroform/methanol (2/1). Once extracted
the fatty acids were trans-methylated with Boron trifluoride methanol 7%
(Sigma-Aldrich, Saint Quentin Fallavier, France). The methyl esters of
plasma fatty acids were analyzed by gas chromatography (Auto Sampling
8410 Gas Chromatograph 3900; Varian, Les Ulis, France) coupled to flame
ionization detector (FID) on an Econo-Cap EC-WAX capillary column (30-m,
0.32-mm internal diameter, 0.25-μm Film, ref 19654, ALLTECH Associates
Inc, Templemars, France), as described [25].
A standard mixture was used to identify each FA methyl ester in
samples, the results were expressed as the abundance of each FA relative
to the total FA (%) (S2 Table), and total fatty acid were quantified using heptadecanoic acid (C17:0, margaric acid) as an internal standard.
Cell culture and incubations
Cells were cultured under standard conditions (moist atmosphere of 5% CO2
at 37°C) in Dulbecco’s Minimum Essential Medium for HaCaT human
keratinocytes (the cell line was obtained directly from the Cell Lines
Service, Batch # 300493–2417) [26] and in RPMI-1640 medium for U937 human leukemic monocytes (the cell line was obtained directly from ATCC® CRL-1593.3TM) [27].
Both mediums were supplemented with 10% fetal calf serum (FCS), 2mM
L-glutamine, 50 IU/ml penicillin and 50 IU/ml streptomycin.
Macrophage and HaCaT seeding
U937 cells were differentiated into macrophages using phorbol myristate acetate (Sigma-Aldrich P8139) at 16 ng/ml for 48h [28].
Once attached to the flask bottom, the cells were scraped, counted and
seeded in 96-well microplates at a density of 200.000 cells per well
(1.106 cells/ml) and kept at 37°C for 24h.
70%
confluent HaCat cells were removed by trypsin incubation and seeded in
96-well microplates at a density of 20.000 cells per well (1.105
cells/ml) and kept at 37°C for 24h. The cells were incubated for 15 min
with different concentrations of CIO, olive oil (as negative control)
or PBS (as cell control), then rinsed in PBS and placed in culture
medium at 37°C for a 24h-recovery period before performing tests [29].
Cell viability assessment using the Alamar Blue assay
Alamar
Blue is a cell viability indicator that uses the reducing power of
living cells to convert resazurin to the fluorescent molecule,
resorufin. Resazurin is a nontoxic, cell permeable compound that is blue
in color and virtually non fluorescent. Upon entering cells, resazurin
is reduced to resorufin, which produces very bright red fluorescence.
Viable cells continuously convert resazurin to resorufin, thereby
generating a quantitative measure of viability using spectrofluorimetry.
We measured cell viability based on the protocol described by Dutot et al. [30].
The cells were incubated with Alamar blue at 0.01mg/ml (Life Technology
DAL1035) in 2.5% FCS culture medium at 3700B0030C for 6h before
performing fluorescent measurement using Safire microplate reader (Tecan
12901300076, λexc = 535 nm, λem = 600 nm).
Wound healing assay
Wound healing assay was adapted from Buonomo’s method [31,32].
HaCaT cells were seeded into 6-well culture plates in 10% FCS culture
medium and kept at 37°C for 72 h. Then, culture medium was removed and
confluent cells were rinsed with PBS and incubated during 24h in culture
medium without FCS. After 24h, confluent cells were wounded with a
pipette tip by manual scratch. Medium was removed, cells were rinsed in
PBS and plates were placed on graduated pattern before taking pictures
of the wound using a Nikon Coolpix camera. PBS was removed and cells
were incubated with CIO, olive oil (as negative control) or PBS (as cell
control) for 15 min followed by 24h-recovery period in 2.5% FCS culture
medium. At D1 (+24h) each wound was taking in picture using the same
method described above. The same three areas per well were compared at
D0 and D1 to determine the percentage of wound closure. ImageJ (1.48v)
was used to analyze pictures. Briefly, wound areas were delimited using
freehand selection tool, then we used measurement tool to edit the data
window that lists the area in μm2 for each wound.
ß-defensin 2 release measurement
The
release of β-defensin 2 in cell supernatants was determined by ELISA
(Phoenix Pharmaceutical: EK-072-37). 24h after CIO exposure or olive oil
exposure (as control), cell supernatants were harvested and stored at
-20°C until β-defensin 2 measurements. The quantity of released
β-defensin 2 was measured according to the manufacturer’s instructions.
Oilogramme
We adapted the dilution method technique [33]
in order to determine the minimal inhibitory concentration (MIC) of
vegetable oils. This new technique based on oil/water emulsions and
convenient to assess MIC with vegetable oils was named oilogramme.
Preparation of dishes:
Tubes containing 18ml of Mueller-Hinton agar (Biomérieux) were
autoclaved and allowed to cool at 50°C in water bath. 2ml of sterile
water, olive oil or CIO were added to the tubes. Tubes containing 2ml of
oil were emulsified, and then serially diluted in MH medium and water
to obtain emulsified MH medium containing 0.001 to 2% final oil
concentrations. Finally, emulsified-oil-containing media were poured on
the dish.
Bacterial strains: Bacterial organisms used were reference and clinical strains choose mostly among species implicated in skin infections (S3–S6 Tables).
Inoculum preparation for MIC test:
Strains were culture overnight on Trycase soy agar (Biomérieux) at
37°C. Then, colonies were transferred into Mueller-Hinton broth
(Biomérieux) and incubated overnight at 37°C. Overnight cultures of
about 1.5 Mc Farland unit (about 2.108 CFU/ml) were used as initial suspensions then, diluted in sterile water as to obtain a concentration of 106−107 CFU/ml. These 106−107
CFU/ml suspensions were transferred in a 96-well culture microplate for
inoculation. Inoculation pattern for each bacteria strain was
represented in S1 Fig.
Inoculation and incubation of the medium:
Agar dishes were spot inoculated, using a multipoint inoculator system
(Multipointelite: SCAN400), with 1μl suspension containing 103−104 CFU, which is the inoculum usually recommended for MIC determination with the agar dilution method [34].
Inoculation was done from the lowest to the highest concentration
plates. Inoculated agar plates were allowed to stand at room temperature
until the inoculum spot were completely absorbed and then incubated at
37°C overnight.
Result interpretation: MIC
represents the lowest concentration of an antimicrobial agent that will
inhibit the visible growth of a microorganism after overnight
incubation. Here, we considered the presence of more than three colonies
per spot as a positive growth.
For oilogramme assays performed on Propionibacterium strains:
we proceeded as described above except for two steps. We used Wilkins
Chalgren agar (Oxoid) and bacteria were incubated at 37°C for 48h in
anaerobic conditions before oilogramme reading.
Bioautographic investigations
Bioautography
is a technique used to detect the antibacterial components present in
an extract. This technique consists in separating extract components on
Thin Layer Chromatography (TLC), pouring agar medium on TLC in bacteria
dishes, spraying bacteria suspension on the agar medium and visualizing
compounds responsible of bacteria growth inhibition [35,36].
TLC (11 cm x 11 cm) were loaded with 0.5, 1 or 2μl of CIO then allowed
for migration using dichloromethane/ethyl acetate (90:10, V/V) as
eluent. Then, separated bands where visualized under UV light (254 and
365 nm). The solvent was completely evaporated from the TLC at room
temperature for 48h before performing antibacterial tests. In order to
determine which band(s) was/were responsible for CIO antibacterial
activity, TLC were placed in bacteria dishes and Muller-Hinton agar
medium was poured to allow bacterial cultures on solid medium. The
inoculum of Staphylococcus aureus was spread over the entire
surface of the agar plate by swabbing and culture was incubated
overnight at 37°C. Then cultures were observed to localize bacterial
growth inhibition areas.
Statistical analysis
All
experiments were conducted in triplicates and values are expressed as
mean ± standard deviation. CIO were compared to olive oil as control to
perform statistical analysis. For all experiments, olive oil has no
effect compared to PBS incubation (data not shown) except in the wound
healing assay, where olive oil induced a statistically significant
effect on wound closure compare to PBS (the percentage of wound closure
was 45±3% with olive oil and 26±2% with PBS). Statistical analysis was
performed using one way ANOVA and results were compared by Student
t-test at a 5% significance level.
Results
Determination of CIO nontoxic concentrations for in vitro experiments
Because CIO could contain toxic agents, we first investigated CIO cytotoxicity. We calculated LC50 as an indicator of CIO toxicity and LC20 as an indicator of CIO maximal nontoxic concentration usable in vitro.
After 15 min of incubation with CIO at the indicated concentrations
(0–30%) and a 24h-recovery period, CIO was found to affect HaCaT cell
viability in a concentration-dependent manner (Fig 1A–1E). The percentage of cell viability was calculated based on a comparison with cells incubated with olive oil. Three CIO (Fig 1F) exhibited comparable LC50 (CIO1: 11.4%, CIO3 and CIO4:12.6%), CIO2 and CIO5 present the lowest and the highest LC50
respectively (CIO2: 18.7% and CIO5:7.3%). These data suggested that the
level of cytotoxic compounds was not the same among tested CIO: CIO5
appears as the most cytotoxic, CIO2 as the less cytotoxic and CIO1,3 and
CIO4 present an intermediary level of toxicity on HaCaT cells. Based on
LC20 (CIO1,4: 7.6%, CIO2:11.6%, CIO3: 8.6% and CIO5: 2.7%)
as maximal nontoxic concentration usable with HaCaT cells, we decided to
study potent CIO therapeutic properties between 0 and 2% in further
experiments.
Cytotoxic effect of CIO on a human keratinocyte cell line: HaCaT cells.
CIO promotes wound healing in HaCaT keratinocyte cells
We
evaluated CIO wound healing stimulating activity on HaCaT cells using
the scratch assay. Scratches were realized on confluent HaCat cells 24h
after CIO exposure at three concentrations (0.01, 0.1 and 1% diluted in
olive oil). At 0.1%, the five CIO induced a statistically significant
effect on wound closure compared to control (Fig 2).
CIO1-4 exhibited their highest wound closure rate at 0.1% (CIO1: 93±4%,
CIO2: 95±6%, CIO3: 68±5% and CIO4: 81±4%) and CIO5 exhibited its
highest wound closure rate at 0.01% (CIO5: 62±6%) compared to control
(Olive oil: 45±3%). These data show that CIO1 and CIO2 exhibit higher
activity on keratinocyte wound closure than the other CIO. In all cases,
CIO needed concentrations to exhibit healing properties are 27 to 76
lower than concentrations presenting cytotoxic effects on keratinocyte
cells, supporting CIO as a safe topical agent.
CIO effect on wound healing.
CIO increase β-defensin 2 release, an antimicrobial peptide active against Gram- bacteria
ß-defensin
2 is an antimicrobial peptide produced by various cells implicated in
innate immune response such as macrophages, and is notably active
against Gram- bacteria [37].
To investigate CIO implication in innate immune response, we assessed
its capacity to induce β-defensin 2 release by U937 derivative
macrophage cells. Macrophages were preincubated with CIO at the
indicated concentrations for 15 min. Supernatants were collected 24h
after CIO exposure to determine β-defensin 2 release. As seen in Fig 3B,
at 0.1% CIO4 is the only one to induce a statistically significant
increase in β-defensin 2 release compared to control (CIO4:
114.51±0.79%). At 1% (Fig 3B)
four of the tested CIO induce a statistically significant increase in
β-defensin 2 release compared to control (CIO1: 107.85±0.02%, CIO2:
109.44±0.62%, CIO4: 115.68±1.03%, CIO5: 118.07±0.77%, olive oil:
100.00±0.85%). Our data indicate that CIO4 and CIO5 are the highest
inducer of β-defensin 2 release in U937 derivative macrophage cells.
CIO effect on β-defensin 2 release by U937 derivative macrophages.
CIO exhibits high antibacterial activity against bacteria involved in skin infections such as multi-drug resistant Staphylococcus aureus strain
To
evaluate the antibacterial properties of CIO, we adapted the dilution
method to determine the Minimal Inhibitory Concentration (MIC) values
with vegetable oils; this new technique was named oilogramme. A
preliminary screening, using oilogramme with 2% CIO showed no activity
against twenty five strains of Gram—bacteria representing twenty species
(S3 Table).
Then, for further investigations, a panel of forty nine aerobic
bacterial strains (forty six Gram+ representing twenty species and three
Gram-), mostly implicated in skin diseases, and including multidrug
resistant strains, was tested against 0.001 to 2% CIO concentrations
using oilogramme (S4 and S5 Tables). Fig 4
shows oilogramme at four oil concentrations (0.4, 0.05, 0.025 and
0.005%), olive oil (used as control) did not inhibit bacterial growth
whatever the tested strain. The three tested aerobic Gram- bacteria
species were Achromobacter xylosoxidans denitrificans, Achromobacter xylosoxidans xylosoxidans and Pseudomonas aeruginosa.
The only bacteria that grew when incubated with 0.4% CIO were the Gram-
strains, while none of the Gram+ strains exhibited growth at this
concentration. These data suggest that the concentrations of CIO we used
here were poorly active against Gram- bacteria. Concerning aerobic
Gram+ bacteria, we tested notably Staphylococcus aureus largely involved in nosocomial and skin infections, Bacillus cereus associated to wound infections in postsurgical patients and cutaneous infections subsequent to trauma, Staphylococcus epidermidis and Staphylococcus haemolyticus responsible for catheter associated infections and Corynebacterium minutissimum implicated in erythrasma. At a concentration of 0.4% all Gram+ bacteria were inhibited in CIO1-5 plates (Fig 5A–5E). These data show that CIO1-5 at 0.4% was active against all studied Gram+ bacteria including a multi-drug resistant Staphylococcus aureus strain (S4 and S6
Tables, strain CRBIP21.21). The MIC value profile for Gram+ bacteria
(min and max MIC values per strain) for each CIO was represented Fig 5.
Gram+ MIC value range was 0.01 to 0.1% for CIO1, 0.01 to 0.2% for CIO2
and CIO4, 0.025 to 0.2% for CIO3 and 0.001 to 0.4% for CIO5 (Fig 5).
Our results show that CIO1 is the most active oil against Gram+
bacteria. Moreover, all the tested CIO against Gram+ bacteria species
present MIC value similar or lower than ofloxacin (S4 Table)
suggesting that CIO could be topically used for prevention or treatment
of Gram+ skin infections. It is interesting to note that the tested
multi-drug resistant Staphylococcus aureus strain is more sensitive to CIO than ofloxacin (0.025% for CIO1 versus 6.400% for ofloxacin) (S4 Table).
In this context, CIO appears as a promising source to develop new
antibiotics notably to fight multi-drug resistant bacteria implicated in
skin infections.
Oilogramme.
CIO antibacterial activity against Gram+ bacteria.
CIO exhibits high antibacterial activity against bacterial strains involved in acne
We also used the oilogramme to test CIO antibacterial activity against twenty three stains of anaerobic Gram+ bacteria, e.g. Propionibacterium acnes and Propionibacterium granulosum, both involved in acne (S5 Table). Fig 5F
shows that the five tested CIO were highly active against these
strains. The MIC value range was 0.01 to 0.025% for CIO1, 0.01 to 0.1%
for CIO2, 0.05 to 0.5% for CIO3, 0.05 to 0.1% for CIO4 and 0.01 to 0.5%
for CIO5. These results show that CIO1 is the most active oil against
both Propionibacterium species. In an interesting manner all the tested CIO against Propionibacterium species present MIC value similar or lower than ofloxacin (S5 Table). These data strongly suggest that CIO could be topically used in the treatment of acne.
CIO presents high selectivity index against Gram+ bacteria
Finally,
we calculated the specific index (SI) also called Therapeutic index for
all the bacteria strains tested against CIO1-5 (Fig 6). SI values were calculated by dividing cytotoxicity LC50 values by the MIC values in the same units (SI = LC50/MIC).
SI is considered as a measure of potential efficacy versus adverse
effects, i.e. the higher the SI is for an extract, the more likely it is
that the activity is not due to a general toxic compound. An SI>1
for an extract increases the likelihood that its toxic activity is not
dependent on the antibacterial compounds [38].
For all the tested CIO the SI values were comprised between 73 and
1260. This high therapeutic index value means that CIO may be used for
treatment of infections with very low toxicity under controlled
conditions.
MIC (%) and specific activity index (SI) of CIO1-5 against 25 bacterial strains calculated by dividing cytotoxicity (LC50) by MIC.
Spotting of CIO antibacterial components by bioautography
To
detect the polarity of antibacterial components present in CIO, we used
bioautography, which consists in separating CIO compounds on Thin Layer
Chromatography (TLC). In a first time, we loaded three TLC with 0.5, 1
and 2μl of CIO1-5. After TLC migration in appropriate solvent, ten major
bands were visualized by UV light (Fig 7A–7F). CIO2, CIO3 and CIO4 exhibited similar profiles compare to CIO1 and CIO5 (Fig 7B). This variation observed in CIO composition seems to depend on CIO geographic origin. Then we used Staphylococcus aureus (CIP 4.83, S4 Table) culture as bacteria spray to localize bacterial growth inhibition areas. Fig 7G–7I
showed that for all the tested CIO the most polar band appearing in
blue under UV at 365 nm provided an inhibition of the bacteria growth at
0.5, 1 and 2μl. Because band n°1 overlaps bands n°2 and n°3 we
performed a new TLC by loading 2μl of a single CIO to better separate
the different components (Fig 7J and 7K). This TLC was used to perform bioautography and Fig 7J–7L showed that band n°1 was indeed responsible for Staphylococcus aureus growth inhibition.
CIO bioautography against Staphylococcus aureus.
Resin extracted from CIO is responsible of its antibacterial activity
As described by Pocidalo et al,
CIO healing properties on animal experimental burns depended on its
resin (21). We therefore hypothesized that others CIO pharmacological
properties such as antibacterial activity could also result from it.
Thus, initially, we separate resin from fatty acids of CIO1-5 following
extraction process previously described by Petard et al (17).
Then we loaded two distinct TLC, one with 0.5μl of CIO1-5 resins and the
other with 0.5μl of CIO1-5 fatty acids. After migration in appropriate
solvent, TLCs were visualized by UV light presented on Fig 8A, 8B, 8D and 8E. We then used Staphylococcus aureus (CIP 4.83, S4 Table) culture on TLCs to localize bacterial growth inhibition areas. Fig 8C
showed that for all the resins tested, the most polar band appearing in
blue under UV at 365 nm provided an inhibition of the bacteria growth
while no inhibition of bacteria growth was observed with fatty acids (Fig 8F). This result indicates that antibacterial activities observed for CIO1-5 were due to its resin.
CIO resin and fatty acid bioautography against Staphylococcus aureus.
Discussion
Due
to antibiotic resistance increase and poor access to treatment in some
tropical areas, new therapeutics accelerating wound closure and at the
same time preventing infections present a great interest (1). Efforts
are needed to find new antibiotics, easy to produce locally and coming
from cheap and renewable sources. Along the seashores and islands of the
Indian and Pacific Oceans, CIO is traditionally topically used to treat
a wide range of skin injuries from burn and infected wounds to skin
disease such as acne and psoriasis. These wound healing and antibiotic
properties make CIO a valuable candidate as alternative therapeutic
strategy to treat infected wounds especially in tropical areas. Using
five CIO that come from Indonesia, Tahiti, Fiji islands and
New-Caledonia we attempted to evaluate their cytotoxicity, wound healing
and antibacterial properties.
We first investigated CIO cytotoxicity to determine nontoxic concentrations usable in vitro.
Then, we investigate CIO wound closure ability on keratinocyte cells.
Our results showed that all of the tested CIO accelerate keratinocyte
wound healing, CIO1 and CIO2 being the most regenerative oils (Fig 9). These results strengthened the wound healing potential of traditionally used CIO.
Schematic representation of the pharmacological activities of the five studied CIO.
As
described earlier, other properties of CIO may participate in the wound
healing process such as elimination of pathogens in infected wounds.
CIO contains palmitic and oleic acids (S2 Table) both known to increase β-defensin 2 production and release [39,40].
This fact should explain that the oily components of CIO such as fatty
acids may have another role than being only a natural excipient of
bioactive components but also by elicitation of β-defensin. β-defensin 2
is an antimicrobial peptide implicate in skin immunity originally
isolated from psoriatic skins [41].
This peptide is produced by various cells implicated in innate immune
response such as keratinocyte and macrophage cells and play an important
antimicrobial role in host defense against cutaneous pathogens [37]. Interestingly β-defensin 2 presents a dose dependent microbicidal effect starting in vitro at concentrations from 1 to 3μM against Gram- bacteria (Pseudomonas aeruginosa, Escherichia coli) and yeasts (Candida albicans) [37].
These data suggest that a small increase in β-defensin 2 release could
be sufficient to improve host defense against cutaneous pathogens. We
showed that CIO increases β-defensin 2 release with CIO4 and CIO5 being
the highest inducers (Fig 9). If this result is confirmed in vivo,
it would mean that CIO could indirectly participate to the elimination
of Gram- bacteria and yeast in skin infections by stimulating innate
immune defenses.
In another experiment, we tested CIO
capacity to directly inhibit bacteria growth. To address the
antibacterial properties of CIO, we used the oilogramme procedure to
determine the MIC values with vegetable oils. This new technique based
on oil/water emulsion doesn’t require the use of organic solvents such
as ethanol or DMSO both known to exhibit cytotoxic effects on living
cells [42] which could interfere with MIC determination [43].
Our data showed that CIO1-5 are poorly active against Gram- bacteria
strains and highly active against all tested Gram+ bacteria strains with
similar or lower MIC values in comparison with ofloxacin (S4 and S5 Tables). Moreover our results indicate that CIO1 is the most active oil against Gram+ bacteria (Fig 9).
It has frequently been stated that plant extracts are more active
against Gram+ bacteria than against Gram- bacteria. This may be
attributed to the cell walls of Gram- bacteria less permeable to
antimicrobial compounds [44].
A previous study showed that CIO presents UV-absorption property [24]. In this we have demonstrated CIO wound healing and antibacterial activity against Propionibacterium acnes and Propionibacterium granulosum,
both involved in acne. Acne is one of the most common skin diseases,
affecting more than 45 million individuals in the United States. It is
estimated that nearly 20 percent of all visits to dermatologists are
related to the treatment of acne [45]. Interestingly, zinc topically used in the treatment of acne, presents a MIC value against Propionibacterium acnes around of 0.26 to 0.51% [46],
whereas the MIC value we observed for CIO1 is around 0.01%. Taken
together, CIO activities present a great interest in the treatment of
this skin disease and making CIO a valuable candidate to develop new
drugs in the treatment of acne.
In recent years, the
scientific community underlined that antibacterial drug development was
not sufficient to address the problems posed by antibiotic resistance
among pathogen bacteria [47].
In our study we showed that compare to ofloxacin CIO are highly active
against a multi-drug resistant strain of a species which is largely
involved in nosocomial and skin infections: Staphylococcus aureus. Furthermore, It is interesting to note that MIC of fucidic acid, topically used for Staphylococcus aureus skin infection, is around 0.1% [48],
whereas the MIC value observed for CIO1 is around 0.023%. In this
context, CIO appears as a promising source to develop new antibiotics
notably to fight multi-drug resistant bacteria involved in skin
infections.
In order to underscore the components responsible for CIO antibacterial property, we performed bioautography against Staphylococcus aureus.
Our data showed that in the five tested CIO, the same band was
responsible of CIO antibacterial activity. Moreover, we showed that this
band was indeed contained specifically in CIO resin and not in the
fatty acid part. Our results suggest that the band presenting the
inhibition could contain coumarins due to its blue fluorescence under UV
[49] and could be in accordance with the antibacterial properties against Staphylococcus aureus observed for four coumarins isolated from the nuts of Calophyllum inophyllum (calaustralin, calophyllolide, inophyllum C, inophyllum E) [23].
Moreover, coumarins were described as water soluble, blue fluorescent
dyes under UV light and they can be qualitatively revealed using Thin
Layer Chromatography (TLC) [49].
Then, we calculated the specific index (SI) for all the bacteria
strains tested against CIO1-5. For all of them the SI values were higher
than 1. These results suggest that CIO could represent a potential safe
source of raw medicine material for clinical treatment of wound
infections.
In an interesting manner, we have shown
that the needed concentrations of CIO to inhibit bacteria growth are
lower than the concentrations needed to promote in vitro wound
healing. These wound healing and antibiotic properties make CIO a
valuable candidate in the treatment of infected wounds. Besides we
observed differences in the level of CIO pharmacological activity
depending on their origin (Fig 9).
For example, we showed that wound healing on keratinocyte cells was
quite similar in CIO3 and CIO4 but lower in CIO5 and higher in CIO1 and
CIO2 (Fig 9)
suggesting variations in CIO composition. Indeed, we observed
variations of composition revealed on TLC with the five CIO and revealed
by fatty acids analysis (S2 Table).
These differences could result from oil extraction processes, genetic
factors as well as environmental influences. Finding of tamanolides
which is a new class of pyranocoumarins from French Polynesian CIO [50],
and not yet reported from the other CIO geographical origins supports
this suggestion. Chemical variability of leaf bioactive components of Calophyllum inophyllum had been also shown at a geographical scale of French Polynesia [51] which may results from the above mentioned factors and should be expected in CIO composition throughout Oceania region.
Further
investigations will be needed to establish and understand the
variations of CIO composition from different origins and how these
variations can influence pharmacological properties of the oil.
Moreover, characterization of CIO phytochemical composition will be
essential to understand CIO antibacterial and regenerative activities
and to further decorticate underneath molecular mechanisms of action.
Conclusions
This
study was conducted to evaluate the cytotoxicity, wound healing and
antibacterial properties of five CIO traditionally used to treat
infected wounds across Oceania. Using human cell and bacteria cultures,
we highlighted the pharmacological effects of CIO, proposed as a wound
healing and antimicrobial agent. We observed differences in the
pharmacological activities of the CIO tested, depending on their origin
and probably their variable compositions. We showed that concentration
of CIO needed to exhibit therapeutic effects are lower than
concentrations exhibiting cytotoxic effects in vitro
substantiating CIO for their safe topically use in infected wounds and
skin diseases such as acne. For the first time, this study provides
support for traditional uses of CIO in the wound healing process
particularly for infected wounds.
Supporting Information
S1 Fig
Aerobic Gram-negative and Gram-positive bacteria repartition on the plates.
Each number correspond to bacteria species described in S3 Table.
(PDF)
Click here for additional data file.(26K, pdf)
S1 Table
References, geographic origins and characteristics of CIO.
(PDF)
Click here for additional data file.(115K, pdf)
S3 Table
Gram-negative bacterial strains tested in preliminary assay.
(PDF)
Click here for additional data file.(335K, pdf)
S4 Table
Aerobic Gram-negative and Gram-positive bacteria tested against CIO.
(PDF)
Click here for additional data file.(1.0M, pdf)
S5 Table
Anaerobic Gram-positive bacteria tested against CIO.
(PDF)
Click here for additional data file.(535K, pdf)
S6 Table
Bacterial strains resistant to antibiotics.
(PDF)
Click here for additional data file.(262K, pdf)
Acknowledgments
We
thank F. Duveau, M. Dutot, E. Olivier, A. Wakx and the staff of the
Laboratoire Ecosystème Intestinal, Probiotiques, Antibiotiques for
technical advice and helpful discussions. We are grateful to J. Bennett,
J-C Bobbia, J-L. Delubriat, A. Rannou and O. Touboul for providing Calophyllum inophyllum oils and to N. Bourgeois-Nicolaos for providing Enterococcus
strains N489, N487, N490, N491 and N492. This work was supported by the
Laboratoire Chimie-Toxicologie Analytique et Cellulaire and by the
Fonds Pacifique (project CALO-PS).
Funding Statement
This work was supported by the Laboratoire Chimie-Toxicologie Analytique et Cellulaire and by the Fonds Pacifique (project CALO-PS).Data Availability
All relevant data are within the paper and its Supporting Information files.
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