J. Biomedical Science and Engineering, 2010, 3, 758-762 JBiSE
doi:10.4236/jbise.2010.38101 Published Online August 2010 (http://www.SciRP.org/journal/jbise/).
Published Online August 2010 in SciRes. http:// www. scirp. org/journal/jbise
Antimicrobial activity of the autochthonous compound Enoxil
Lucian Lupaşcu1, Valeriu Rudic1,2, Viorica Cotos3, Tudor Lupaşcu4
1Institute of Microbiology and Biotechnology of the ASM, Chişinău, Moldavia;
2Academy of Sciences of Moldova (ASM), Chişinău, Moldavia;
3Hospital of Contagious Diseases “T. Ciorba”, Chişinău, Moldavia;
4Institute of Chemistry of the ASM, Chişinău, Moldavia.
Received 24 February 2010; revised 18 March 2010; accepted 31 March 2010.
ABSTRACT
The paper presents data about the antimicrobial ac-
tivity of the autochthonous compound of taninic
source Enoxil. The minimal inhibitory and bacteri-
cidal/fungicidal concentrations were established for
some skin and wound infectious agents. It was estab-
lished, based on Pseudomonas bacteria model, that
the Enoxil compound suppress the activity of some
important enzymes—a phenomenon that leads to the
increase of bacterial sensibility to many tested anti-
biotics.
Keywords: Bacteria; Compound; Tannins; Wound;
Infection
1. INTRODUCTION
Skin and wound infections, although more characteristic
for countries with low economic level and low hygiene
standards [1], have lately become a serious concern for
highly socio-economically developed countries, as well
[2-5]. Of great concern are also the intrahospital plague
infections, as they complicate the recovering process,
produce anxiety, increase patient’s discomfort and may
lead to death, being at the same time a serious threat to
the medical worker [5,6]. The economical aspects of
infections are also considerable [7]. The most frequent
microorganisms in the derma or the purulent wound are
the gram-positive bacteria Staphylococcus aureus and
the gram-negative bacteria Escherichia coli, Pseudomo-
nas aeruginosa [8]. Special attention is paid to pseudo-
monadic infections as complications of combustions,
traumas, tattoo procedures, cellulites, decubital chronic
ulcers, foliculitis [9-11].
One of the traditional methods of fighting infections is
the use of antibiotics. However, a concerning phenome-
non is observed lately in the entire world, including the
Republic of Moldova—resistance of microorganisms to
these compounds [12,13]. A permanent and, often, abu-
sive treatment with antibiotics had as a result the selec-
tion of new virulences, aggressive and severe, with a
high genetic resistance potential. This situation directs
the specialists in the field towards the elaboration of
antimicrobial compounds on the basis of novel action
mechanisms [14]. Albeit the use of tannin extracts from
plants for the treatment of various derma and plagues
infections has been known for a long time, these don’t
exhibit the expected effect for many microorganisms
[15-17]. At the moment, the number of efficient medici-
nal compounds, elaborated on the basis of tannin com-
pounds is quite low, mostly due to the insolubility of
many of them in aqueous or alcoholic solutions. The
capacity of phenolic (tannin) extracts to disinfect and
rapidly treat wounds is based on two fundamental phe-
nomena—antimicrobial and antioxidant activities, de-
termined by a series of mechanisms: capturing of iron
ions from the substrate, thus depriving microorganisms
of compounds necessary for their physiological activity;
their binding to microbial proteins and formation of
complexes; capturing of free radicals; absorption of
oxygen radicals; inhibition of low density lipoproteins
oxidation [18]. It is known that grape seeds are a rich
source of so called enotannins–condensed tannins which
represent a wide variety of natural substances with
polyphenolic structure, remarkable due to the high con-
tent of proanthocyanidins [19]. There are enotannins in
industrial quantities in the Republic of Moldova. Taking
into account that many tannins with antimicrobial prop-
erties are insoluble in water, as well as the growing mi-
crobial resistance to vegetal tannins, of great potential
are researches regarding the possibility of structural
modification of enotannins, increase of their oxidation
number, in order to increase their efficiency and to use
them for the treatment of skin and plague infections.
The objective of present researches was to elucidate
the antimicrobial activity of the autochthonous com-

L. Lupaşcu et al. / J. Biomedical Science and Engineering 3 (2010) 758-762 759
pound Enoxil of tanninic origin.
2. MATERIALS AND METHODS
Research was carried out at the Department of Microbi-
ology, Immunology and Virusology of the State Univer-
sity of Medicine and Pharmaceutics “N.Testemiteanu”.
The autochthonous compound Enoxil served as the
antimicrobial remedy, obtained by hydro-solubilization
of enotannins using chemical and physico-chemical
procedures [20]. As microbial cultures, served several
pathogens with severe implications in many contagious
diseases—bacteria Staphylococcus aureus, Escherichia
coli, Salmonella abony, P.aeruginosa, Proteus vulgaris
and the fungus Candida albi cans.
In order to determine the level of activity of the tested
compound, the values of minimal inhibiting concentra-
tion (MIC) and the minimal bactericidal/fungicidal con-
centration (MBC/MFC) were used [21]. As test indices
of the action of Enoxil on the P.aeruginosa bacteria,
served a range of important biochemical parameters –
indices of bacterial viability and pathogenicity: synthesis
of cytochrome oxidase, citrate reductase, haemolysins,
pyocyanin, odour presence, and as the index of sensibil-
ity towards antibiotics—zone of inhibition (mm) of the
culture, specific to each antibiotic. The sensibility of the
P.aeruginosa bacteria was determined using the me-
thod of antibiotics diffusion in gelose from roundels
[22] that contained the following antibiotics homolo-
gated in the Republic of Moldova: cloramfenicol (30 mkg),
pefloxaciline (5 mkg), erythromycin (15 mkg), cefu-
roxim (30 mkg), cefoxitin (30 mkg), cefalotine (30 mkg),
piperacillin (30 mkg), imipenem (10 mkg), ciprofloxacin
(5 mkg), tobramycin (10 mkg), gentamicin (10 mkg),
tetracycline (30 mkg). The standardized nutritive me-
dium Miuller-Hinton was used for testing. The boxes
with bacteria and antibiotics were maintained at 37°C,
for 24 hours, with a subsequent determination of the
result of antibioticogram.
The acute toxicity research during the enteral admini-
stration of the Enoxil compound was made on mouses
and white rats. It was established that the 500, 1000 and
2000 mg/kg body weight doses do not provoke modifi-
cations in the animals behaviour and neither their death.
The cronical toxicity was established by the administra-
tion of the Enoxil in 100 and 300 mg/kg body weight
doses at the male rats during 30 days.
Data were statistically analysed using the soft package
STATISTICA 7.
3. RESULTS AND DISCUSSIONS
During the research was established that the animals that
took the 300 mg/kg dose were more adinamic and were
dead in 5-21 days time, but those administrated the 100
mg/kg dose survived. At macroscopical examination of
the internal organs were not attested any pathological
modifications. After undertaken the cronical toxicity
study was established that the Enoxil compound in the
100 mg/kg dose do not modify esentially the level of
creatinin and cholesterol.
Investigations on the action of Enoxil compound on
bacteria E.coli, S.abony, S.aureus, P.vulgaris, P.aerugi-
nosa and the fungus C.albicans resulted in the determi-
nation of the MIC (Figure 1(a)) and the minimal bacte-
ricidal/fungicidal concentration (MBC/MFC) (Figure 1(b)).
Thus, Enoxil presents antibacterial, as well as antifungal
properties at relatively low concentrations. The minimal
(a)
(b)
Figure 1. (a) Minimal inhibiting concentration and
(b) Bactericidal/fungicidal of Enoxil for several mi-
croorganisms. On the hexagonal perimeter: 1–E.coli,
2–S.abony, 3–S.aureus, 4–P.vulgaris, 5–P. aeruginosa,
6–C.albicans. On the vertical–concentrations, %.

760 L. Lupaşcu et al. / J. Biomedical Science and Engineering 3 (2010) 758-762
inhibiting concentration of Enoxil for the tested bacteria
was shown to be in the range 0.15-0.3%, while the bac-
tericidal one—in the range 0.3-0.6%. For the fungus
C.albicans the minimal inhibiting concentration equals
to 0.3%, while the fungicidal one—to 0.6%.
The values MIC and MBC/MFC for the microorgan-
isms taken into the study presented differences of an
order of magnitude, except for the E.coli bacteria, when
these indexes coincided. As a result of the correlation
analysis, it was determined that the degree of depend-
ence (r) between MIC and MBC/MFC is significant and
positive, equal to 0.66. Regression analysis, which has a
predictive value and represents the mathematical relation
of the dependence, demonstrated that its equation is: y =
0.0923 + 0.3385 x (p ≤ 0.05).
Data presented in Table 1 shows that under the action
of Enoxil at the determined static concentration (0.15%)
took place the suppression of activities of oxidases, cit-
rate reductases and haemolysin. At the same time, the
bacterial culture didn’t produce any pyocyanin and in-
dole, and it didn’t develop any specific odours. In these
conditions, the maintenance of the mobility is a proof of
bacteria viability, which reveals the correctitude of the
test. Thus, the static concentration of Enoxil produces
significant perturbations at the biochemical level inside
the bacterial cell, which probably significantly dimin-
ishes the capacity of substrates decomposition, as well as
its pathogenic potential.
Treatment of the bacterial culture with Enoxil in-
creased the sensitivity (S) for the majority of investi-
gated antibiotics, with manifested resistance (R) only for
erythromycin and cefalotine (Table 2).
In the case of tetracycline, a reaction of medium sen-
sitivity was observed (RS). In the case of blank, the cul-
ture manifested sensitivity only for pefloxaciline and
imipenem, while in the case of using Enoxil, the sensi-
bility was exhibited towards the majority of antibiotics,
except for erythromycin and cefalotine. It should me
mentioned that Enoxil introduced sensibility into the
bacteria towards several antibiotics considered inactive
for P.aeruginosa, for example cefoxitin. The analysis of
the similitude degree of the compounds using the clus-
terian method of dendrograms construction, demon-
strated that the tested antibiotics were distributed in big
clusters (Figure 2(a)), quite different, which reveals that
Table 1. Influence of the static concentration of Enoxil on
several biochemical and functional indices of the P.aeruginosa
bacterium.
Indicator Blank (+/–) Enoxil (+/–)
Oxidase + –
Citrate reductase + –
Haemolysin + –
Indole – –
Pyocyanin + –
Odour + –
Mobility + +
Table 2. Influence of the static concentration of Enoxil on the sensibility of the culture P. aerugino sa towards
several antibiotics.
Untreated culture (blank) Treated culture
Nr. Antibiotic Inhibition zon e, mm Reaction Inhibition zone, mm Reaction
1 Cloramfenicol 0.0 R 26.0 ± 0.6* S
2 Pefloxaciline 14.0 ± 0.6 R 26.3 ± 0.3* S
3 Erythromycin 0.0 R 13.0 ± 0.6* R
4 Cefuroxim 0.0 R 20.0 ± 1.2* S
5 Cefoxitin 0.0 R 22.0 ± 0.6* S
6 Cefalotine 0.0 R 0.0 R
7 Piperacillin 0.0 R 20.0 ± 1.2* S
8 Imipenem 30.0 ± 1.2 S 29.3 ± 1.5* S
9 Ciprofloxacin 0.0 R 24.0 ± 0.6* S
10 Tobramycin 0.0 R 17.0 ± 1.2* S
11 Gentamicin 0.0 R 16.0 ± 0.6* S
12 Tetracycline 0.0 R 19.0 ± 0.7* R-S
* - difference from the blank with statistic support at p < 0.05.

L. Lupaşcu et al. / J. Biomedical Science and Engineering 3 (2010) 758-762 761
(a)
(b)
Figure 2. Clusterian analysis ((a)–construction of the dendro-
gram; (b)–method of k-means) of antibiotics on the basis of the
capacity of suppression of p.aeruginosa strain treated with
enoxil (0.2%). (a) on the vertical: 1–cloramfenicol, 2–peflox-
aciline, 3–erythromycin, 4–cefuroxim, 5–cefoxitin, 6–cefa-
lotine, 7–piperacillin, 8–imipenem, 9–ciprofloxacin, 10–to-
bramycin, 11–gentamicin, 12–tetracycline. on the horizontal: 0
-40–euclidian distances. (b) on the vertical: 1, 2, 3–clusters of
antibiotics. On the horizontal: 1, 2, 3–repeats.
the bacterial stem treated with Enoxil has different reac-
tion towards antibiotics. A significantly different cluster
was presented by the compound 6 – cefalotine (one of
the most efficient antibiotics in pseudomonadic infec-
tions), for which the stem manifested the greatest resis-
tance (inhibition zone: 0.0 mm). The lowest is the ag-
gregation degree of clusters, the greatest is antibiotics
similitude, on the basis of the inhibition zone (for exam-
ple, cloramfenicol–1 and pefloxaciline–2). As opposed
to the method of dendrograms construction, which is an
agglomeration-iterative method and performs objects
classification (antibiotics) on the basis of various aggre-
gation levels, the method k-means of clusterian analysis,
non-iterative, classifies objects in clusters, determined
by the researcher, on the basis of possible effects: low,
medium, high, etc. and presents opportunities of high
precision [23].
The classification (repartition) of antibiotics on the
basis of the action on the P.aeruginosa strain, treated
with Enoxil evidenced the existence of clusters with pro-
nounced differences (Figure 2(b)). Through the exami-
nation of members (antibiotics) of the 3 clusters, it was
determined that cluster 1 was formed by cefalotine alone,
for which P.aeruginosa manifested the highest resistance,
which was confirmed by the analysis of the repartition
dendrogram. The cluster 2, where the antibiotics eryth-
romycin, cefuroxim, piperacillin, tobramycin, genta-
micin, tetracycline were distributed, is characterized by
their capacity to produce a reaction of medium sensibil-
ity, while the cluster 3 – antibiotics cloramfenicol, pe-
floxaciline, cefoxitin, imipenem, ciprofloxacin – a reac-
tion of strong sensibility.
Thus, if we make abstraction of the antibiotic imipe-
nem, for which the strain presented sensibility in the
blank as well, it may be observed that Enoxil induced a
reaction of strong sensibility for the majority of antibi-
otics, among which 3 antibiotics – cloramfenicol, pe-
floxaciline and cefalotine are not used for the treatment
of pseudomonadic infections [24], i.e. are non-specific.
4. CONCLUSIONS
The values MIC and MBC/MFC of Enoxil for the inves-
tigated microorganisms presented differences of an order
of magnitude, except for the bacterium E.coli, for which
these indices coincided. It was found that there is a high
and positive correlation for MIC/MBC during treatment
with Enoxil, which makes microorganisms behaviour
predictive.
On the model of the P.aeruginosa bacteria it was de-
termined that several of the biochemical mechanisms of
suppression of growth and development by Enoxil con-
sist in the inhibition of synthesis of such enzymes, im-
portant for the bacterial organism viability, as oxidases,
citrate reductases, haemolysins, as well as pyocyanin.
This phenomenon influenced most directly the sensibil-
ity of the bacteria towards a series of antibiotics used in
modern medicine. The compound Enoxil in bactericidal
concentrations (MBC–0.3%) may be considered of great
potential in the treatment of pseudomonadic infections.
5. ACKNOWLEDGEMENTS
The authors are very grateful to Prof. V. Gonciar from the Department
of Pharmacology from the State University of Medicine and Pharmacy
“N.Testemitanu” who carried out the research on the toxicity of the
Enoxil compound.

762 L. Lupaşcu et al. / J. Biomedical Science and Engineering 3 (2010) 758-762
REFERENCES
[1] Dogra, S. and Kumar, B. (2003) Epidemiology of skin
diseases in school children: A study from Northern India.
Pediatric Dermatology, 20(6), 470-473.
[2] Collier, M. (2002) Wound-bed management: Key princi-
ples for practice. Professional Nurse, 18(4), 221-225.
[3] Cooper, R. (2002) Wound microbiology: Past, present,
and future. British Journal of Nursing, 11(22), 4-6.
[4] Miller, M. (2001) Wound infection unravelled. Journal of
Community Nursing, 15(3), 31-33.
[5] Nosocomial Infection National Surveillance Service (2002)
Surveillance of surgical site infection in English hospitals:
A national surveillance and quality improvement pro-
gramme. Health Protection Agency, London.
[6] Singh, N.P., Goyal, R., Manchanda, V., et al. (2003)
Changing trends in bacteriology of burns in the burns
unit. Burns, 29(2), 129-132.
[7] Prisacari, V. and Paraschiv, A. (2008) Contrubutions in
the epidemiological surveillance systems optimization
within nosocomial septico-purulent infections of surgi-
cal profile. Epidemiology and Microbiology. Mat. Con-
gress VI of Hygienists, Epidemiologists and Microbi-
ologists from the Republic of Moldova, Chişinău, 2,
22-25.
[8] Rodgers, G., Mortensen, J., Fisher, M., et al. (2000) Pre-
dictors of infectious complications after burn injuries in
children. Pediatric Infectious Disease Journal, 19(10),
990-995.
[9] Gang, R.K., Bang, R.L., Sanyal, S.C., et al. (1999) Pseu-
domonas aeruginosa septicaemia in burns. Burns, 25(7),
611-616.
[10] Kehheth, T. (2008) Pseudomonas aeruginosa. http://www.
textbookofbacteriology.net/pseudomonas.html
[11] Rozhavin, M.A. (1983) Some biological properties of the
P.aeruginosa melanin. Journal of Microbiology, in Rus-
sian, 1, 45-47.
[12] Valencia, I., Kirsner, R. and Kerdel, F. (2004) Micro-
biologic evaluation of skin wounds: Alarming trend to-
ward antibiotic resistance in an inpatient dermatology
service during a 10-year period. Journal of the Ameri-
can Academy of Dermatology, 50(6), 845-849.
[13] Visnevchi, L. and Bradu, V. (2008) Antibiotical microor-
ganisms resistance modification, tested in the microbi-
ological laboratory of PRMC Ungheni. Epidemiology
and Microbiology. Mat. Congress VI of Hygienists, Epi-
demiologists and Microbiologists from the Republic of
Moldova, Chişinău, 2, 264-265.
[14] Schwaber, M.J., Cosgrove, S.E., Gold, H.S., et al. (2004)
Fluoroquinolones protective against cephalosporin resis-
tance in gram-negative nosocomial pathogens. http://www.
medscape.com/viewarticle/466484
[15] Asres, K., Mazumder, A. and Bucar, F. (2006) Antibacte-
rial and antifungal activities of extracts of combretum
molle. Ethiopian Medical Journal, 44(3), 269-277.
[16] Santos, S.C. and Mello, J.C.P. (2004) Taninos. Farma-
cognosia da Planta ao Medicamento, in Portuguese, 5th
Edition, Porto Alegre, Florianópolis, 615-656.
[17] Timbola, A.K., Szpoganicz, B., Branco, A., et al. (2002)
A new flavonol from leaves of Eugenia jambolana. Fi-
toterapia, 73(2), 174-176.
[18] Chung, K., Lu, Z. and Chou, M. (1998) Mechanism of
inhibition of tannic acid and related compounds on the
growth of intestinal bacteria. Food and Chemical Toxi-
cology, 36(12), 1053-1060.
[19] Kulciţki, V., Vlad, P., Duca, G., et al. (2007) Investigation
of grape seed proantocyanidins. Chemistry Journal of
Moldova, 2(1), 36-50.
[20] Lupascu, T. and Lupascu, L. (2006) The obtaining pro-
cedure of the watersoluble enotannins. Patent Nr. 3125.
MD. BOPI: 8.
[21] Andrews, J.M. (2001) Determination of minimum in-
hibitory concentrations. Journal of Antimicrobial Che-
motherapy, 48(1), 5-16.
[22] National Committee for Clinical Laboratory Standards
(NCCLS) (2002) Performance standards for antimicro-
bial susceptibility testing. 12th Informational Supplement
M100–S12 NCCLS, Wayne.
[23] Halafian, A.A. (2008) Modern statistical methods of
medical research. In Russian, LKI Publishers, Moscow.
[24] Matcovschi, C., Procopisin, V. and Parii, B. (2006) Phar-
macotherapeutical guide. Central Printing, Chişinău.