1. INTRODUCTION:
Lipids are present ubiquitously in nature, and are known to
exhibit potential anti-bacterial activities against bacteria (Gram-negative and
Gram-positive), viruses, fungi and parasites (Nakatsuji et al., 2009;
Schlievert et al., 1992). Fatty acids (FAs) and 1-monoglycerides (MGs) have an
established history to inhibit/kill pathogens after they get access into human
body. The in vitro antibacterial actions exhibited by FAs are
characteristically broad spectrum with their potencies analogous to many
natural antimicrobial peptides (Georgel et al. 2005).
The main targets of antibacterial lipids and Fatty acids are
bacterial cell membrane and some of essential cellular process taking place in
cell. The surfactant properties of FAs may cause formation of
transient/permanent pores leading to exudation of essential cellular contents.
The high concentration of fatty acids may also lead to solubilisation of the
cell membrane and causing the release of membrane proteins or larger sections
of the lipid bilayers (Boyaval et al., 1995; Wojtczak and Więckowski,
1999). Other synergistic process contributing the bacterial lysis could be (1)
the inhibition of enzyme activity, (2) meddling of essential nutrient uptake
and (3) formation of toxic peroxidation/ autooxidation. Lipids such as
fatty alcohols, free fatty acids and monoglycerides of fatty acids are known to
be potent antimicrobial/microbicidal agents in vitro and to kill enveloped viruses, gram-positive and
gram-negative bacteria and fungi on contact (Thormar and Hilmarsson 2007, Singh
et al., 2014, Singh et al., 2015b). However, no pharmaceutical products containing lipids as active
compounds have as yet been approved for clinical use as prophylactic or
therapeutic drugs against bacterial and viral infections, even after the proven
antimicrobial activities of lipids (Thormar and Hilmarsson 2007, Singh et
al., 2014, Singh et al., 2015b).
Apparently, the great success of chemotherapy using synthetic antibiotics
against bacterial and fungal infections and nucleoside analogues against viral
infection has discouraged researchers and the pharmaceutical industry in making
serious efforts to develop drug containing simple natural compounds (Thormar and
Hilmarsson 2007, Singh et al., 2014, Singh et al., 2015b). The present study aimed to screen and
optimized the levels of lipids and surfactants that holds an innate
antibacterial activity against gram positive and gram negative bacteria and
which can be transformed into nanoemulsions.
2. MATERIALS AND
METHODS:
E. coli (MTCC-1678), P. aeruginosa (MTCC-647), S.
aureus (MTCC-3160) and B. subtilis
(MTCC-430) were obtained from the Microbial Type Culture Collection IMTECH
(Institute of Microbial Technology, Chandigarh, Punjab, India). They were used
throughout the experiment by maintaining and reviving every twenty days in
nutrient agar media at pH 7.0. Labrasol and Cremophor® RH40 were gifted from
Gattefosse (St-Priest, France). Oleyl amine was purchased
from Fluka (Steinheim, the Netherlands).
2.1 Screening of lipids/surfactants on the zone of
inhibition studies:
Solid medium diffusion procedure using wells in plates was used to
determine the antibacterial activity of explored lipids/surfactants charge
inducers against gram negative (E.coli
and P. aeruginosa) and gram positive
(S. aureus and B. subtilis) bacteria. Petri plates (90 mm diameter) (Tarson,
India) were prepared by pouring 25 mL sterile nutrient agar (NA) and allowed to
solidify. Plates were dried and 500 μL of an inoculum suspension (~105-106
cfu/mL) was poured and uniformly spread. After inoculums absorption by agar,
wells were made using sterile tubes (diameter 5 mm) which were filled with 100
μL of 5 % w/v of each excipients. The plates were incubated in incubator
at 37 ºC for 24 hr. The negative control used in this method was sterile water
for injection. The inhibition zones were measured in millimetres using vernier
callipers. The excipients which were insoluble in sterile water for injection
were made dispersed by incorporating Cremophor RH40 (5 % w/w). It is worth
mentioning that the Cremophor RH40 had no anti-bacterial activities against the
selected bacterial strains at 5 % w/w.
2.2 Anti-bacterial
studies of excipients:
2.2.1
Optimization of Capmul MCM C8 and labrasol concentration using colony count
method:
The optimization of Capmul MCM C8 and Labrasol
concentration was designed according to the method reported by Zhang et al.,
2010 and Singh et al., 2015a with some modifications. The 5.0 mL bacterial
cultures (~105-106
cfu/mL) were added to 5.0 mL of various
concentrations (0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 % w/v) of capmul
MCM C8 and labrasol in a sterile test tube and incubated for 60 min at 37 oC.
Control bacterial samples (E. coli, P. aeruginosa, S. aureus and B. subtilis)
were prepared in a similar method by adding 5.0 mL of Millipore water. Next,
0.5 mL of samples were taken from each tube and added to sterilize molten
Nutrient Agar (NA) plates. The NA plates were then incubated at 37 oC
for 24 hrs and then counted for viable colonies. The experiment was performed
in triplicate for each set of conditions. The percent inhibition of microbial
count at different concentrations was calculated by number of colonies found on
treated plates divided by that of the control blank.
2.2.2 Optimization of combined effects of Capmul
MCM C8 and labrasol using colony count method:
After the initial screening and
concentration optimization of capmul MCM C8 and labrasol, the stock solution of
1:1 ratio of capmul MCM C8 and labrasol (Smix) were prepared. The Smix was than
suitably diluted with sterile water for injection to get the working
concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 and 4 % w/v. The 5 mL
of these working concentrations were mixed with 5 mL of bacterial cultures (~105-106
cfu/mL) and incubated for 60 min at 37 oC. Control bacterial samples (E. coli, P. aeruginosa, S. aureus and B. subtilis) were prepared in a similar method by adding 5.0 mL of
Millipore water. The percent inhibition of microbial count at different
concentrations was calculated by number of colonies found on treated plates
divided by that of the control blank.
2.2.3 Optimization of oleyl amine concentration
using colony count method:
The Smix (1:1 ratio of capmul MCM C8 and
labrasol) prepared in section were suitably diluted to get the concentration of
1.0 % w/v. The diluted solutions were admixed with oleylamine to get the final
working oleyl concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 % w/v.
The admixture was vortexed for 30 minutes to ensure complete dispersion of
oleylamine. The 5 mL of these admixtures were mixed with 5 mL of bacterial
cultures (~105-106 cfu/mL)
and incubated for 60 min at 37 oC. Control
bacterial samples (E. coli, P. aeruginosa, S. aureus and B. subtilis)
were prepared in a similar method by adding 5.0 mL of Millipore water. The
percent inhibition of microbial count at different concentrations of time was
calculated by number of colonies found on treated plates divided by that of the
control blank.
3.0 Optimization
of Cremophor RH 40 concentrations by determining mean particle size:
The Smix (1:1 ratio of capmul MCM C8 and
labrasol) prepared were suitably diluted to get the concentration of 1.0 % w/v.
The diluted solutions were admixed with cremophor RH 40 to get the final
working cremophor RH40 concentrations of 0.25, 0.375, 0.5, 0.75, 1.0, 1.5 and 2
% w/v. The admixture was vortexed for 30 minutes to ensure complete adsorption
of cremophor RH40. Similarly, 1.0% w/v of 1:1 ratio of Smix was admixed with
0.5 % w/v of oleyl amine followed by adding cremophor RH 40 to get the same
working concentrations of surfactant. All the prepared formulations were than
subjected to determination of mean particle size using Zetasizer
(Nano ZS; Malvern Instruments, UK) with He–Ne red laser, 4.0 mW, 633 nm;
temperature, 25 ◦C;
refractive index, 1.333; or with adjustment if needed. All measurements were in
triplicate done using disposable polystyrene cuvettes (Malvern Instruments,
UK).
4.0 RESULTS AND DISCUSSIONS:
4.1 Screening of lipids/surfactants on the zone of
inhibition studies:
Zone of inhibition (mm) exhibited by different oils, surfactants/
co-surfactants against selected bacterial strains are tabulated in Table 1.
The in vitro results revealed the maximum zone of inhibition in
capmul MCM C8 amongst various explored lipids. The zone of inhibition was found
to be 16.0 ± 0.5, 13.3 ± 0.6,
20.1 ± 0.5 and 18.4 ± 0.7 against E. coli, P. aeruginosa, S. aureus and B. subtilis, respectively. Accordingly, labrasol showed maximum zone amongst various
explored surfactants/co-surfactants. The zone of inhibition was found to be 14.3 ± 1.2, 11.5 ± 0.4, 15.2 ± 0.7 and 14.5 ± 0.3
against E.
coli, P.
aeruginosa, S.
aureus and B. subtilis,
respectively. Furthermore, Gram positive bacteria (S. aureus and B. subtilis)
were found to be more susceptible to various explored excipients as compared to
Gram-negative bacteria (E. coli, P. aeruginosa).
In all classes of capmul, caprylic acid (C8:0) content is more
than 80% as compared to other fatty acids such as capric acid (C10:0).
Similarly, miglyol classes contain more than 50% caprylic acid (C8:0). Gelucire
44/14 contains lauric cid (C12:0) as major component (44.7%) and caprylic acid
nearly 7.29%. Labrasol is mixture of mono-, di-and triglycerides that contains
mainly caprylocaproyl macrogol-8-glycerides. Labrasol exclusively contains
50-80% caprylic (C8:0) and 20-30 % capric acid (C10:0). Hence, the inherent
anti-bacterial activity may be attributed to caprylic acid present lipids and
surfactants. Labrasol apart from eliciting anti-bacterial activity is also a
potent P-pg inhibitor. This may lead to enhanced oral bioavailability of
anti-bacterial drugs (if loaded) and may cause reduced drug resistance. From
the above study, capmul MCM C8 and labrasol was selected as lipid and
surfactant phases, respectively. These selected excipients are considered as
generally regarded as safe category (GRAS).
4.2 Optimization of Capmul MCM C8 and labrasol concentration using colony count method:
The
selected lipid (Capmul MCM C8) and surfactant (labrasol) were further subjected
to susceptibility test against all the four bacterial strains (E. coli, P.
aeruginosa, S. aureus and B. subtilis) by colony count method. Figure 1 demonstrates the percent
viable colonies remaining when treated with varied concentration of capmul MCM
C8. No any viable colonies of gram negative bacteria (E. coli, P.
aeruginosa) were detected
at 4.0 and 5.0 % w/v, respectively. Gram positive bacteria (S. aureus and B.
subtilis) were found to
be more susceptible as compared to gram negative bacteria with complete
inhibition at 3.0 % w/v of capmul MCM C8 (Figure
1).
Figure
1 Percent
viable colonies of gram positive (S. aureus and
B. subtilis) and gram negative (E.
coli, P. aeruginosa) remaining when treated with varied concentrations of
capmul MCM C8 (% w/v).
Similarly, Figure 2 illustrates the percent viable colonies
remaining when treated with varied concentration of labrasol. No viable
colonies of gram negative bacteria (E. coli, P. aeruginosa) were detected at 5.0 and 7.0 % w/v,
respectively. In case of Gram positive bacteria (S. aureus and B.
subtilis) no viable
colonies were detected at 5.0 and 6.0 % w/v of labrasol (Figure 2).
Furthermore, the studies demonstrate that the capmul MCM C8 was more
susceptible in killing both gram positive and gram negative bacteria as
compared to labrasol.
Figure
2 Percent viable colonies of gram positive
(S. aureus and B.
subtilis) and gram negative (E.
coli, P. aeruginosa) remaining when treated with varied concentrations of
labrasol (% w/v).
4.3
Optimization of Combined Effects of
Capmul MCM C8 and Labrasol using Colony Count Method:
This study was planned to screen the optimum concentration of Smix
(1:1 ratio of capmul MCM C8 and labrasol) that gives maximum inhibition of all
the four bacterial strains. Figure 3
illustrates the percent viable colonies remaining when treated with varied
concentration of Smix. The inset of Figure
3 illustrates the enlarged portion of the figure to efficiently adjudge the
anti-bacterial effect at lower Smix concentrations. As can be seen from Figure 3, no any viable colonies were
found at ~1.0 % w/v of Smix concentration. Furthermore, the Gram positive
bacteria (S.
aureus and
B. subtilis) were found
to be more susceptible as compared to gram negative bacteria (E. coli, P. aeruginosa) as judged by the slope of killing
curves. Amongst gram negative bacteria, E.
coli was found more susceptible as compared to P. aeruginosa. Similarly, amongst Gram positive bacteria, S.aureus was found to be marginally more
susceptible as compared to B. subtilis (Figure 3).
Figure
3 Percent viable colonies of gram positive (S.
aureus and B. subtilis) and gram negative (E.
coli, P. aeruginosa) remaining when treated with varied concentrations of Smix
(1:1 ratio of capmul MCM C8 and labrasol) (% w/v). The inset shows the enlarged
portion of the figure.
4.4 Optimization of oleylamine (charge inducer)
concentration using colony count method:
The bacterial cell is primarily negatively charged due to anionic biophosphorylated sugar head groups of lipo-polysaccharides
as well as due to negatively charged lipids like cardiolipin. It is therefore
anticipated that cationic charge inducer on lipid droplets may further enhance
the bactericidal effect. In the present study, we have selected oleylamine as possible
charge inducer. The concentration of the charge inducer in the formulation
needs to be optimized. This study was envisioned to screen the optimized levels
of oleylamine in Smix (1:1 ratio of capmul MCM C8: labrasol) by carrying out
anti bacterial studies on all the four selected bacterial strains. Figure 4 illustrates the comparative
evaluation of percent viable colonies remaining when treated with varied
concentration of oleylamine (% w/v). As demonstrated in Figure 4, 0.5 % w/v of oleylamine enabled the complete loss of
viable colonies in all strains of selected bacteria. Furthermore, as discussed previously, the Gram positive bacteria (S. aureus and B.
subtilis) were found to
be more vulnerable as compared to gram negative bacteria (E. coli, P. aeruginosa) as observed by the slope of killing
curves. Amongst gram negative bacteria, E.
coli was found more prone as evaluated against P. aeruginosa. Similarly, amongst Gram positive bacteria, S. aureus was found to be slightly more
vulnerable as compared to B. subtilis (Figure 4).
Figure
4
Percent viable colonies of gram positive (S. aureus and
B. subtilis) and gram-negative (E. coli, P. aeruginosa) remaining when
treated with varied concentrations (% w/v) of oleyamine present in Smix (1:1ratio
of capmul MCM C8 and labrasol).
5.0 Optimization of Cremophor RH 40 concentrations
by determining mean particle size:
The lipidic droplets have to be stabilized to coalescence by
introducing the surfactant which lowers down the interfacial tension existing
between lipid and aqueous phase. In the present study we have selected
Cremophor RH 40 as potential non-ionic surfactant. The non-ionic surfactants
are known to be least toxic as compared to ionic surfactant. Furthermore, this
surfactant belongs to the category of generally recognised as safe category.
The concentration of cremophor RH 40 was optimized by recording the mean
particle size. The surfactant concentration that gave least particle size was
selected as optimized level. Figure 5
illustrates the mean particle size of
Smix (1:1 ratio of capmul MCM C8 and labrasol) obtained at varied
concentrations of cremophor RH 40. The mean particle size was obtained in
presence and absence of oleylamine. The analysis of Figure 5 indicates that the mean particle of Smix in absence of
oleylamine decreased from 300.32 ± 5.64 nm to 58.43 ± 0.34 nm when cremophor RH
40 concentration increased from 0.25 % w/v to 0.75 % w/v. Thereafter, from 1.0
% w/v to 2.0 % w/v, there was insignificant change in mean particle size (10.34
± 0.22 to 11.24 ± 0.32). Similarly, mean particle of Smix in presence of
oleylamine decreased from 358.34 ± 8.32 nm to 110.45 ± 2.23 nm when cremophor
RH 40 concentration increased from 0.25 % w/v to 0.75 % w/v. Thereafter, from
1.0 % w/v to 2.0 % w/v, the mean particle ranged between 54.21 ± 1.34 to 59.32
± 1.43, illustrating insignificant variations. Furthermore, the mean particle
size was found to be at higher side in presence of oleylamine as compared to
formulations which does not contain charge inducer. This might be due to
formation of oleylamine layer onto Smix droplets.
Figure
5
Mean particle size (nm) of Smix (1:1 ratio of capmul MCM C8 and Labrasol) obtained at varied concentrations of
cremophor RH 40. The mean particle size was recorded in presence and absence of
charge inducer.
CONCLUSIONS:
Gram
positive bacteria (S. aureus and B. subtilis) were found to be more
susceptible to various explored excipients as compared to Gram-negative
bacteria (E. coli, P. aeruginosa). In case of Gram positive bacteria (S. aureus and B.
subtilis) no viable
colonies were detected at 5.0 and 6.0 % w/v of labrasol. 0.5 % w/v of oleylamine enabled the complete loss
of viable colonies in all strains of selected bacteria. The mean particle of Smix in absence of
oleylamine decreased from 300.32 ± 5.64 nm to 58.43 ± 0.34 nm when cremophor RH
40 concentration increased from 0.25 % w/v to 0.75 % w/v. Thereafter, from 1.0
% w/v to 2.0 % w/v, there was insignificant change in mean particle size (10.34
± 0.22 to 11.24 ± 0.32). Similarly, mean particle of Smix in presence of
oleylamine decreased from 358.34 ± 8.32 nm to 110.45 ± 2.23 nm when cremophor
RH 40 concentration increased from 0.25 % w/v to 0.75 % w/v. Thereafter, from
1.0 % w/v to 2.0 % w/v, the mean particle ranged between 54.21 ± 1.34 to 59.32
± 1.43, illustrating insignificant variations. The results of the study
indicate that the capmul MCM C8 and labrasol can be selected as potential lipid
and surfactant for the preparation of nanoemulsions which inherits potential
antibacterial activities. The oleyl amine can be used as potential charge
inducer to amplify the antibacterial activities
ACKNOWLEDGEMENT:
Authors
acknowledges University Grant Commission (UGC), Government of India for
providing the financial assistance vide sanction letter number “41-1423/2012
(SR), dated 30 July 2012, to Mrs. Neeru Singh.
6.0
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