Ester-bonded cationic gemini surfactants: Assessment of their cytotoxicity and antimicrobial activity

Article abstract of DOI:10.1016/j.molliq.2016.07.044

We have assessed the biological properties such as cytotoxicity and antimicrobial activity of a series of biocompatible ester-linked cationic gemini surfactants, ethane-1,2-diyl bis(N, N-dimethyl-N-alkylammoniumacetoxy) dichlorides (m-E2-m, m?=?12, 14, 16). The effect of alkyl chain length (12, 14 and 16 carbon atoms) on their antimicrobial activity and cytotoxicity was examined. Antimicrobial activity of m-E2-m, against various prokaryotic and eukaryotic microorganisms, was studied by measuring the diameter of inhibition zone whereas cytotoxicity was evaluated using 3T3-L1 fibroblast cells. Toxic effect against these cells depends upon the type of the target microorganisms, nature of the cells, and hydrophobicity of the molecules. Antimicrobial activity of the gemini surfactant 16-E2-16 is lower than that of its corresponding single-chain counterpart. The gemini surfactants used for the present study (m-E2-m) which have excellent surface properties and much lower cmc values show low toxicity and significant antimicrobial activity.

Full text of DOI:10.1016/j.molliq.2016.07.044

Journal of Molecular Liquids 222 (2016) 390–394
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Ester-bonded cationic gemini surfactants: Assessment of their
cytotoxicity and antimicrobial activity
a
b
Nazish Fatma a, Manorama Panda a, , Kabir-ud-Din , Muheeb Beg
⁎
a
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
b
Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow 226031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We have assessed the biological properties such as cytotoxicity and antimicrobial activity of a series of biocom-
patible ester-linked cationic gemini surfactants, ethane-1,2-diyl bis(N, N-dimethyl-N-alkylammoniumacetoxy)
dichlorides (m-E2-m, m = 12, 14, 16). The effect of alkyl chain length (12, 14 and 16 carbon atoms) on their an-
timicrobial activity and cytotoxicity was examined. Antimicrobial activity of m-E2-m, against various prokaryotic
and eukaryotic microorganisms, was studied by measuring the diameter of inhibition zone whereas cytotoxicity
was evaluated using 3T3-L1 fibroblast cells. Toxic effect against these cells depends upon the type of the target
microorganisms, nature of the cells, and hydrophobicity of the molecules. Antimicrobial activity of the gemini
surfactant 16-E2-16 is lower than that of its corresponding single-chain counterpart. The gemini surfactants
used for the present study (m-E2-m) which have excellent surface properties and much lower cmc values
show low toxicity and significant antimicrobial activity.
Received 2 December 2015
Received in revised form 12 July 2016
Accepted 13 July 2016
Available online 15 July 2016
Keywords:
Ester-bonded gemini surfactants
Critical micelle concentration
Micelle
Cytotoxicity assessment
Antimicrobial activity
© 2016 Published by Elsevier B.V.
1. Introduction
microbial resistance without causing damage on the host cells. The
lower degradability, high toxicity and more expensive preparation of
Gemini surfactants are composed of two hydrophobic chains linked
at or near the head groups by a short, long, rigid or flexible spacer [1].
These amphiphiles are of substantial interest owing to their superior
physicochemical behavior such as excellent surface-active properties,
good surface tension lowering ability at the surface, unusually low crit-
ical micelle concentrations (cmc), high viscoelasticity, higher solubiliza-
tion capacity and good foaming ability in comparison with the
conventional surfactants having one lipophilic part and one hydrophilic
group in the molecule [2]. The superiority of gemini surfactants is found
in almost every field of surfactant applications and, therefore, much at-
tention has been focused on the development of gemini surfactants for
improving their efficiency. These intriguing qualities of geminis along
with the increasing demand for high performance surfactants make
them of special interest for pharmaceutical applications. As compared
to traditional surfactants, applications of gemini surfactants are multi-
fold in cleaning agents and detergents, cosmetics and personal care
products, pharmaceutical, biomedical, biological and in gene transfec-
tion applications [3]. In general, quaternary ammonium salts are widely
used as better disinfectants in medicine, agriculture and industry; but
for more resistant organisms, search of potent antimicrobial agents is
more important. Presently, development of new antibacterial and anti-
fungal agents with novel architecture as well as superior action remains
the important criteria for the chemists for the resolution of increasing
cationic surfactants are the major concerns. To overcome these prob-
lems a number of meaningful steps have been taken to synthesize bio-
compatible gemini surfactants. The insertion of weak or labile bonds
in the spacer part causes the formation of more cleavable, easily degrad-
able gemini surfactants because of which less toxic materials are
discharged during their use. Cationic gemini surfactants with cleavable
ester groups in the hydrophilic parts - the ecofriendly surfactants -
have, therefore, been developed [4–6]. These geminis are a new class
of compounds with low toxicity, good biodegradability, excellent sur-
face properties and are environmentally acceptable. Once used, the sur-
factants are expected to get hydrolyzed and biodegraded with ease.
A series of cationic geminis, m-E2-m (m = 12, 14, 16, is the number
of carbon atoms in the hydrocarbon chain and E2 represents two ester
groups in the spacer part), were synthesized with the intention of de-
veloping eco-friendly amphiphiles whose properties are superior to
the conventional as well as some other gemini surfactants containing al-
kene spacer. The insertion of diester group in m-E2-m geminis incul-
cates very low cmc values than the traditional homologues [7,8].
Bacteria, the prokaryotic cells, are present ubiquitously in the bio-
sphere and form relationships - either beneficial or harmful - with
other organisms. Some chemical compounds have capability to reduce
the growth and metabolism of pathogenic or non-disease-causing mi-
croorganisms. Generally, halogens and their compounds like chlorine,
chloramines, bromine and quaternary ammonium salts are considered
as better disinfectants. Combination of these halogenated compounds
with water results in halo compounds which decompose to give nascent
⁎
Corresponding author.
E-mail address: manoramapanda01@gmail.com (M. Panda).
http://dx.doi.org/10.1016/j.molliq.2016.07.044
0167-7322/© 2016 Published by Elsevier B.V.

N. Fatma et al. / Journal of Molecular Liquids 222 (2016) 390–394
391
oxygen. The nascent oxygen reacts with the enzymes in the cell wall of
microorganism causing the death of microbes. The microbial life is af-
fected by interfacial phenomenon because the cell wall is the most ob-
vious interface through which organisms contact with their
environments. The traditional surfactants adsorb onto negatively
charged surfaces of the substrates exclusively by an ion-exchange
mechanism. The quaternary ammonium compounds (QACs) interact
with the membrane protein of microbes and destruct their structure
[9]. There is a need for new antibacterial agents which would be effec-
tive against these bacteria without causing any harm to the host cells.
In this context, cationic gemini surfactants are known to show excellent
behavior against a broad range of microorganisms. The synthesis and
antimicrobial properties of amphiphiles have been reported by many
workers [10–13].
2.2.2. Cytotoxicity assay (MTT test)
The MTT - based cytotoxicity test was done for the dicationic gemini
surfactants m-E2-m and the conventional cationic surfactant
cetyltrimethylammonium chloride (CTAC) (Fig. 1). 3T3-L1 fibroblast
cell (American Type Culture Collection (ATCC; Manassas, VA, Catalog
number CL-173)) preadipocytes of mouse were maintained in DMEM
containing 10% (v/v) fetal bovine serum and penicillin/streptomycin an-
tibiotics (cDMEM).
The MTT assay was performed on the cells seeded into 96-well
plates at a density of 1 × 104 cells/well in 200 μl in (cDMEM) Dulbecco's
Modified Eagle Medium powder (high glucose medium). After 24 h, the
culture medium was replaced by fresh media containing different con-
centrations of surfactant solutions (500 μM, 100 μM, 10 μM and 1 μM);
the cells were incubated for 24 h at 37 °C and 5% CO2 in CO2 incubator.
Then 25 μl aliquot of MTT (yellow tetrazole), 5 mg/ml in PBS (pH = 7.4),
was added to each well and then incubated again for 4 h at 37 °C.
The supernatant (the MTT solution) was removed carefully and the
insoluble formazan crystals were dissolved in 200 μl/well dimethyl sulf-
oxide (DMSO). Absorbance of the solution was measured by a BMG
FLUOstar Galaxy spectrophotometer (BMG Lab Technologies Ltd.,
Germany) at a wavelength of 544 nm [17,18].
In this study, we have examined the toxicity profile and antimicrobi-
al properties of the diester containing cationic gemini surfactants
(Scheme 1) with varying hydrophobicity towards the biological (fibro-
blast) cells, bacterial and fungal cells.
2. Experimental section
2.1. Materials
2.2.3. Antimicrobial activity
The surfactants 12-E2-12, 14-E2-14 and 16-E2-16 were synthesized
in our lab following the chemical routes reported earlier [6,8,14–16].
Dulbecco's Modified Eagle Medium powder (DMEM) from Gibco
invitrogen (Cat. No.-12100-046, 98%), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) from Sigma (Cat. No. M-
2128, 98%), chloramphenicol (Hi-Media, India), nystatin (Hi-Media,
India) and dimethyl sulfoxide (Sigma-Aldrich, USA, N99%) were used
for the study.
The gemini surfactants were tested for their antimicrobial activity
against bacteria and fungi in the following manner [19].
2.2.3.1. Growing of microorganisms. The bacterial strains were cultured
on a nutrient broth and incubated at 37 °C for 24 h whereas the fungus
strain was cultured on SDB (Sabouraud Dextrose Broth) and incubated
at 37 °C for approximately 18–48 h.
2.2.3.2. Measurements of resistance and susceptibility. Agar well method
was used to determine the antimicrobial activity of the test compounds.
Here 0.1 ml bacterial culture suspension was spread on to nutrient agar
plates uniformly while for fungus SDA (Sabouraud Dextrose Agar)
plates were used. Sterile 8 mm wells (Hi-media Pvt. Ltd) were cut and
loaded with the test compounds. Each plate had one negative control
disc impregnated with the solvent. The antibiotic discs, chlorampheni-
col (30 μg/disc) and nystatin (30 μg/disc), were used, respectively, as
controls for antibacterial and antifungal studies. The antibacterial and
antifungal activities of the surfactants were determined by measuring
the diameters of the inhibition zone (in mm) at minimum inhibitory
concentration (MIC) 400 μg/ml.
2.2. Methods
2.2.1. Synthesis route of the biocompatible gemini surfactants
The gemini surfactants were synthesized by two main steps. First
step involved the preparation of ethane-1,2-diyl bis(chloroacetate). In
a round bottom flask, chloroacetyl chloride (0.22 mol) was added slow-
ly to ethane-1,2-diol (0.1 mol), then the mixture was heated for 8 h at
50 °C in nitrogen atmosphere. The product was washed with saturated
brine (NaCl) solution till complete neutralization; after that it was dis-
solved in diethyl ether, then dried over magnesium sulphate. Needle-
shaped crystals were obtained.
In the second step, the desired gemini was synthesized by heating
ethane-1,2-diyl bis(chloroacetate) with N, N-dimethylalkylamine
(molar ratio = 1:2.1) in ethyl acetate for 10 h. The solvent was removed
under reduced pressure and white crystalline solid of the final product
was obtained. The product was recrystallized in ethyl acetate-ethanol
mixture (5:1) and was obtained in good yield. The purity and structure
confirmation of the gemini surfactants (m-E2-m) were done via some
analytical methods like Silica gel thin layer plate chromatography
(TLC), elemental (C, H, N) analysis, FT-IR, 1H NMR and ESI-MS (+) spec-
troscopy. The presence of no minima in the surface tension versus log
[surfactant] plots was taken as additional evidence regarding the purity
of the surfactants. The synthesis protocol is illustrated in Scheme 2.
Some important properties of m-E2-m are presented in Table 1.
3. Results and discussions
3.1. Cytotoxicity assay (MTT test)
The MTT assay is colorimetric assay and is used to analyze the toxic-
ity of any compound on living cells. This assay determines the reduction
of yellow MTT to dark purple blue color, absorbance of which gives the
proportion of viable cells. Mechanistically, MTT enters into the cells and
passes into the mitochondria where the mitochondrial enzyme succi-
nate dehydrogenase reduces it to insoluble dark purple formazan prod-
uct. Hence, this reduction is found in all viable and metabolically active
cells, the level of activity being a measure of viability of cells. In our ex-
periments, we have used 3T3-L1 fibroblast cell line to assess the toxicity
of three gemini surfactants (m-E2-m, m = 12,14,16). The cytotoxicity
on the 3T3-L1 cells was evaluated by the absorbance versus concentra-
tion plots. All the data are expressed as mean
standard deviations
for three different experiments. From Fig. 1, it is clear that the dimeric
surfactants are appreciably less toxic than the monomeric surfactant,
CTAC. In case of single-chain surfactants, it is expected that the toxicity
increases with longer alkyl chains. However, for the gemini surfactants
it is possible that the toxicity levels decrease with higher alkyl chains
[20,21]. Hydrophobicity and charge density might be responsible for
Scheme 1. Structure of the diester-linked cationic gemini surfactants (m-E2-m, m = 12,
14, 16) used for the present investigation.

392
N. Fatma et al. / Journal of Molecular Liquids 222 (2016) 390–394
Scheme 2. Synthesis path of the biodegradable diester-bonded cationic gemini surfactants (m-E2-m; m = 12, 14, 16).
the variation in toxicity of the compounds. Influence of these parame-
ters is different for various types of cells used for evaluating the toxicity.
Here, the cells in media are taken as control and CTAC is for comparison
of toxicity with the gemini surfactants. It is evident from Fig. 1 that 12-
E2-12 is less toxic up to 100 μM that means about 90% cells are viable
and only 10% cells are dead as compared to control. In case of the con-
ventional surfactant, toxicity was observed at all the concentrations.
For 1 μM of CTAC about 87% cells were found alive as compared to con-
trol. We have used a broad range of concentrations of the geminis and
compared them with the conventional surfactant, CTAC. There is visible
difference in absorbance at the concentrations 1 μM and 10 μM. CTAC
caused around 50% death of the cells at 10 μM, while none of the gem-
inis was showing any toxicity symptom indicated by absorbance. As
we gradually moved towards the higher concentrations, CTAC became
saturated even at 10 μM and no further sign of toxicity was observed
while the compound saturation limit shifted to 100 μM in case of gem-
inis (except 12-E2-12 which causes just 12% toxicity).
statistically significant. 12-E2-12 is extremely statistically significant,
less toxic and is close to control.
3.3. Antimicrobial activity
Cationic surfactants are of much interest because of their
membrane-disruptive and rapid antimicrobial activities [22–24]. These
surfactants are, often, active against a broad spectrum of microbes in-
cluding bacteria and other cells [25]. Due to their high affinity towards
biological membranes, these surfactants show a low selectivity and
may produce damaging effects to a variety of mammalian cells [26–28].
As the time required for control of the growth of microorganisms
with the cationic gemini surfactants is small, it is expected that side ef-
fects in the host might be decreased by the use of easily degradable and
less toxic substances which are subject to hydrolytic degradation. The
products obtained during the hydrolysis process should also be signifi-
cantly less toxic than the parent compound and should constitute nor-
mal metabolites of the host. For example, dilute solutions of the
amphiphile, CTAB (cetyltrimethylammonium bromide), are used in
eye-drop formulations due to its mild anti-bacterial properties; also,
the use of low concentrations of dioctadecyldimethylammonium bro-
mide (DODAB) to reduce bacterial contamination of water has been
suggested [29]. To explore the possible use of degradable cationic gem-
ini surfactants, we studied antimicrobial activity of these ester-linked
cationic gemini surfactants against various prokaryotic and eukaryotic
microorganisms which was evaluated by measuring the diameter of in-
hibition zone (mm) (Table 2) produced by diffusion of the m-E2-m
from the cup into the surrounding medium.
3.2. Statistical analysis
Statistical analysis was carried out by using Student's t-test for
paired data. Experimental data are expressed as means standard er-
rors of the means (SEM) with the help of SPSS 16. The level of signifi-
cance was set at P b 0.05. By conventional criteria for P b 0.001, this
difference is considered to be extremely statistically significant. For
14-E2-14 and 16-E2-16, at 500 μM, 100 μM are considered to be
The cationic surfactants become intercalated into the hydrophobic
interior of the microbial membrane and the cationic polar head groups
participate in electrostatic charge interactions with neighbouring sur-
face structures [18]. In general, antimicrobial activities of these surfac-
tants are dependent on length of the hydrophobic alkyl tail and show
interaction with lipid bilayer structures of cell membranes. Further-
more, these cause leakage of cytoplasmic compounds, indicating that
the plasma membrane is also affected [30,31]. Charge interactions be-
tween quaternary nitrogen groups of m-E2-m and phosphate groups
Table 1
Properties of the diester-linked cationic gemini surfactants.
Surfactant
Molecular formula
Mw (g/mol)
cmc (mM)
12-E2-12
14-E2-14
16-E2-16
C34H70O4N2Cl2
C38H78O4N2Cl2
C42H86O4N2Cl2
641
697
753
0.0016a
0.0014b
0.0013c
a
Ref. [14].
Ref. [15].
Ref. [14].
b
c

N. Fatma et al. / Journal of Molecular Liquids 222 (2016) 390–394
393
Fig. 1. Toxicity profiles of gemini surfactants (m-E2-m; m = 12, 14, 16). The cells were cultured and treated as mentioned in the Experimental section. Absorbance of reduced MTT was
taken at 544 nm. Data are expressed as mean standard deviation of MTT significant at * P b 0.05, ** P b 0.01 and *** P b 0.001 in three independent experiments.
in phospholipids and lipopolysaccharide may lead to complex forma-
tion [18].
of a thick wall containing many layers of peptidoglycan and teichoic
acids [34]. The mechanism of action of compounds on microorganisms
depends on the hydrophobicity and amphiphilic nature of surfactant.
There is an attractive interaction of the hydrophobic chains with the
lipid layers and protein of the cell membrane which facilitates the ad-
sorption on cell surface of microbes. The compounds also showed mod-
erate to good inhibitory results against gram-negative bacteria E. coli,
but no activity was observed for P. aeruginosa. As expected, the gram-
negative bacteria were more resistant than the gram-positive bacteria.
Some gram-negative bacteria were relatively insensitive because their
outer membranes are composed of high lipopolysaccharides and pro-
teins which restrict the entrance of biocides and some hydrophobic-
hydrophilic compounds [5]. Thus, the antibacterial activity of m-E2-m
is higher against gram-positive than against gram-negative bacteria.
Further, antifungal screening reveals that the geminis exhibit moderate
to good inhibition to C. albicans. The fungal membranes are composed of
glucosamine and chitin, which give these cells higher rigidity and resis-
tance for biocide agent. It has been reported that the cationic surfactants
having multi-polar groups show higher antimicrobial activities than the
single headed cationic surfactants because of high charge density of the
head groups [23]. Among the three geminis and the conventional sur-
factants, used in the present investigation, 12-E2-12 showed more po-
tent inhibitory activity against both types of bacteria and the others
showed good inhibition towards C. albicans. Antibacterial activity de-
creases from 12-E2-12 to 16-E2-16.
Antibacterial activity of the cationic amphiphiles was investigated
and the results were compared with those of the nonhydrolyzable sur-
factant, CTAB [32]. The bacterial cell surface is generally negatively
charged, and hence, the cationic surfactants interact with the bacterial
cell wall by electrostatic interaction as well as by the hydrophobic inter-
action. The interaction of cationic m-E2-m might be due to
(i) adsorption on the cell surface of bacteria, and (ii) binding to the cy-
toplasmic membrane via diffusion through the cell wall and disruption
of cytoplasmic membrane resulting in death of the cell. Electrophoretic
measurements have clearly demonstrated that the bacterial cell surface
is usually negatively charged, and hence, the adsorption of cationic am-
phiphiles on the negatively charged cell surface is facilitated by electro-
static interaction, along with the hydrophobic interaction [33].
The cationic geminis were tested in vitro antibacterial activity
against an assortment of two gram-positive bacteria (Staphylococcus au-
reus SA 22, Bacillus subtilis MTCC 121) and two gram-negative bacteria
(Escherichia coli K12, P. aeruginosa). In vitro antifungal activity was test-
ed against a fungal strain, Candida albicans IOA-109. Minimum inhibito-
ry concentrations (MIC) of the tested compounds were 400 μg/ml. The
geminis exerted significant inhibitory activity against the growth of
the tested bacterial and fungal strains. The data (Table 2) reveal that
the compounds have significant influence on antibacterial profile of
gram-positive bacteria. Because of the difference in structure and com-
position of bacterial cell wall, they show different activity towards the
tested compounds. The gram-positive bacterial cell wall is composed
4. Conclusions
Cytotoxicity and antimicrobial activity of three homologues of ester-
bonded cationic gemini surfactants are presented. The lower toxicity
profile of diester bonded gemini surfactants opens the new approach
in pharmaceutical (drug solubilization and drug targeting) and biomed-
ical applications. The geminis show antimicrobial activity and act as soft
drug. The compounds were screened in vitro antibacterial activity
against the representative panel of two gram-positive and two gram-
negative bacteria, and also tested for their inhibitory action against
one strain of fungus. The geminis exerted significant inhibitory activity
against growth of the tested bacterial and fungal strains. The data reveal
that the compounds have significant influence on antibacterial profile of
gram-positive bacteria. Findings of this investigation can provide
Table 2
In vitro antibacterial and antifungal activities of the gemini surfactants.
Surfactants
Gram positive
B. subtilis S. aureus E. coli K12 P. aeruginosa C. albicans
Diameter of inhibition zone in mm at 400 μg/ml
Gram negative
Fungus
12-E2-12
14-E2-14
16-E2-16
Chloramphenicol 24
Nystatin
16
14
13
19
16
11
26
–
10
11
11
25
–
–
–
–
23
–
21
18
20
–
–
20
–, No activity detected.

394
N. Fatma et al. / Journal of Molecular Liquids 222 (2016) 390–394
[9] W.G. Salt, D. Wiseman, J. Pharm. Pharmacol. 22 (1970) 261.
[10] F.M. Menger, C.A. Littau, J. Am. Chem. Soc. 115 (1993) 10083.
[11] L. Perez, J.L. Torres, A. Manresa, C. Solans, M.R. Infante, Langmuir 12 (1996) 5296.
[12] F. Devinsky, I. Lacko, D. Mlynarcik, V. Racansky, Tenside Deterg. 22 (1985) 10.
[13] M.S. Sheikh, A.J. Khanam, R.H. Matto, J. Surfactant Deterg. 16 (2013) 503.
[14] N. Fatma, W.H. Ansari, M. Panda, Kabir-ud-Din, Z. Phys. Chem. 227 (2013) 133.
[15] N. Fatma, M. Panda, W.H. Ansari, Kabir-ud-din, J. Mol. Liq. 211 (2015) 247.
[16] N. Fatma, W.H. Ansari, M. Panda, Kabir-ud-Din, J. Surfactant Deterg. 16 (2013) 609.
[17] A. Bhadani, S. Singh, Langmuir 25 (2009) 11703.
valuable information for selection of surfactants for microbiocidal ac-
tion; results of the present study can be used, beneficially, to have a
clear idea on the antibacterial activity and cytotoxicity of the surfactant
systems.
Acknowledgements
[18] M. Ding, X. He, Z. Wang, J. Li, H. Tan, H. Deng, Q. Fu, Q. Gu, Biomaterials 32 (2011)
9515.
[19] J. Haldar, P. Kondaiah, S. Bhattacharya, J. Med. Chem. 48 (2005) 3823.
[20] Y. Wang, E.F. Marques, J. Phys. Chem. B 110 (2006) 1151.
[21] A. Bhadani, S. Singh, Langmuir 27 (2011) 14033.
[22] M.L. Ancelin, H.J. Vial, Antimicrob. Agents Chemother. 29 (1986) 814.
[23] B. Isomaa, H. Hagerstrand, G. Paatero, A.C. Engblom, Biochim. Biophys. Acta 860
(1986) 510.
[24] S. Ishikawa, Y. Matsumura, K. Katoh-Kubo, T. Tsuchido, J. Appl. Microbiol. 93 (2002)
302.
Manorama Panda acknowledges financial assistance under the De-
partment of Science and Technology, Government of India (Project No.
SR/WOS-A/CS-46/2007), Nazish Fatma is thankful to the University
Grants Commission (India) for fellowship, Kabir-ud-Din is thankful to
UGC for awarding Faculty Fellowship under its BSR Program and
Muheeb Beg acknowledges the Division of Pharmacology, Central
Drug Research Institute, Lucknow (India), for facilities.
[25] M. Rucka, M. Oswiecimska, S. Witek, Environ. Prot. Eng. 9 (1983) 25.
[26] G.I.L. Paatero, D.L. Brown, P.D. Waterhouse, Cell. Mol. Biol. 32 (1986) 79.
[27] K. Yamauchi, M. Tomita, T.J. Giehl, R.T. Ellison III, Infect. Immun. 61 (1993) 719.
[28] G. Ronsin, A.J. Kirby, S. Rittenhouse, G. Woodnutt, P. Camilleri, J. Chem. Soc. Perkin
Trans. 2 (2002) 1302.
[29] W.H. Martindale, The Extra Pharmacopeia, Pharmaceutical Press, London, 1994.
[30] M. Lindstedt, S. Allenmark, R.A. Thompson, L. Edebo, Antimicrob. Agents Chemother.
34 (1990) 1949.
References
[1] F.M. Menger, C.A. Littau, J. Am. Chem. Soc. 113 (1991) 1451–1452.
[2] R. Zana, J. Xia (Eds.), Gemini Surfactants: Synthesis, Interfacial and Solution-phase
Behavior, and Applications, CRC, USA, 2003.
[3] P. Camilleri, A. Kremer, A.J. Edwards, K.H. Jennings, O. Jenkins, I. Marshall, C.
McGregor, W. Neville, S.Q. Rice, R.J. Smith, M.J. Wilkinson, A.J. Kirby, J. Chem. Soc.
Chem. Commun. (2000) 1253.
[31] B.M. El-Sadek, Adv. Appl. Sci. Res. 2 (2011) 363.
[4] T. Banno, K. Kawada, S. Matsumura, J. Surfactant Deterg. 13 (2010) 387.
[5] L. Perez, M.T. Garcia, I. Ribosa, M.P. Vinardell, A. Manresa, M.R. Infante, Environ.
Toxicol. Chem. 21 (2002) 1279.
[6] G. Zhinong, T. Shuxin, Z. Qi, Z. Yu, L. Bo, G. Yushu, H. Li, T. Xiaoyan, Wuhan J. Natural
Sci. 13 (2008) 227.
[32] A. Shirai, T. Maeda, H. Nagamune, H. Matsuki, S. Kaneshina, H. Kourai, Eur. J. Med.
Chem. 40 (2005) 113.
[33] A.T. Poortinga, R. Bos, W. Norde, H.J. Busscher, Surf. Sci. Rep. 47 (2002) 1.
[34] P. Hugenholtz, Genome Biol. 3 (2002) (Reviews 0003.1).
[7] Kabir-ud-Din, M.S. Sheikh, A.A. Dar, J. Phys. Chem. B 114 (2010) 6023.
[8] W.H. Ansari, N. Fatma, M. Panda, Kabir-ud-Din, Soft Matter 9 (2013) 1478.