Hindawi Publishing Corporation

Evidence-Based Complementary and Alternative Medicine

Volume 2013, Article ID 614501, 5 pages
http://dx.doi.org/10.1155/2013/614501

Research Article
Antimicrobial Activity of Lippia Species from the Brazilian
Semiarid Region Traditionally Used as Antiseptic and
Anti-Infective Agents
Cristiana da Purificao Pinto,1 Velize Dias Rodrigues,1 Fernanda da Purificao Pinto,1
Renata da Purificao Pinto,1 Ana Paula Trovatti Uetanabaro,2
Carla Santos Ribeiro Pinheiro,3 Suzana Ferreira Magalhes Gadea,3
Tnia Regina dos Santos Silva,3 and Anglica Maria Lucchese1
1

Laboratorio de Qumica de Produtos Naturais e Bioativos, Departamento de Ciencias Exatas,

Universidade Estadual de Feira de Santana, Avenida Transnordestina S/N, Bairro Novo Horizonte, Campus Universitario,
44036-900 Feira de Santana, BA, Brazil
2
Laboratorio de Microbiologia da Agroindustria, Universidade Estadual de Santa Cruz, Campus Soane Nazare de Andrade,
Km 16 Rodovia Ilheus-Itabuna, 45662-900 Ilheus, BA, Brazil
3
Departamento de Ciencias Biologicas, Universidade Estadual de Feira de Santana, Avenida Transnordestina S/N,
Bairro Novo Horizonte, Campus Universitario, 44036-900 Feira de Santana, BA, Brazil
Correspondence should be addressed to Angelica Maria Lucchese; [email protected]
Received 15 July 2013; Accepted 7 August 2013
Academic Editor: Vincenzo De Feo
Copyright 2013 Cristiana da Purificacao Pinto et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Lippia origanoides Kunth, Lippia alnifolia Schauer, and Lippia thymoides Martius and Schauer are shrubs used in the traditional
Brazilian medicine as antiseptics, as well as in the treatment of infectious diseases. This study was designed to investigate the
antibacterial and antifungal activities of the methanolic extracts of these species, as new potential sources of antimicrobial drugs.
The antimicrobial activity of methanolic extracts was investigated against resistant yeasts and bacteria by agar disk diffusion.
Then, the MIC determination of the most active species and its fractions in hexane, dichloromethane, ethyl acetate, and water
was performed. By the agar diffusion assay, all species were active against at least two microorganisms, giving evidence to support
their use in the popular medicine. L. origanoides leaves exhibited the widest antimicrobial action, inhibiting the growth of two
Gram-positive bacteria and two yeasts; this activity was also confirmed by the MIC evaluation. The fractionation of L. origanoides
crude extracts improved the activity in spectrum and intensity. The results obtained in this study indicate that L. origanoides may
be a promising alternative in the treatment of bacterial and fungal infections and in the seeking of new antimicrobial drugs.

1. Introduction
The development and spread of antimicrobial resistance is a
worldwide concern, due to the negative impacts on public
health [1]. Therefore, there is a growing need for novel drugs
against bacterial, fungal, and viral infections. The traditional
knowledge on the therapeutic potential of plants has been
attracting scientific interest seeking new ways to control and
treat many diseases caused by microorganisms [24].

The genus Lippia has a great number of medicinal species,

such as L. origanoides Kunth, L. alnifolia Schauer, and L.
thymoides Martius and Schauer, which are frequently used
in folk medicine for the treatment of microbial diseases [5].
In Brazil, L. origanoides is popularly known as salva-deMarajo and alecrim dAngola. The leaf infusions are used
as a general antiseptic for the mouth, throat, and wounds,
for the treatment of baby colic, diarrhea, indigestion, flatus,
heartburn, nausea, vaginal discharges, menstrual complaints,

2
and fever [5, 6]. L. alnifolia is also known as alecrimdo-mato, pedrecio, and alecrim-de-vaqueiro. The leaves
are employed as a topic antiseptic against dermatitis and
dandruff, as well as an oral antiseptic and in vaginal infections
[7, 8]. Aerial parts of L. thymoides, known as alecrim-domato, are used in the treatment of skin infections [7].
Although the traditional use of these plants, studies supporting the antibiotic activity of L. alnifolia and L. thymoides
are not found in the literature. Antimicrobial activity has
been reported to L. origanoides, though it was related to the
leaf essential oils [7, 911]. Therefore, a bioprospecting study
was designed to investigate the antimicrobial activities of L.
alnifolia, L. origanoides, and L. thymoides extracts from the
Brazilian semiarid region against bacteria and yeasts, in order
to select the most active species.

2. Materials and Methods

2.1. Plant Material. Aerial parts of L. alnifolia (LA), L. origanoides (LO), and L. thymoides (LT) were collected in
October 2006, in the city of Rio de Contas, BA, Brazil.
Samples were identified by Dr. Tania Regina dos Santos Silva,
from Universidade Estadual de Feira de Santana. Voucher
specimens were deposited at the UEFS herbarium, under the
registry numbers HUEFS 112586, HUEFS 112591, and HUEFS
112597, respectively.
2.2. Extracts Preparation. The air dried and powdered stems,
leaves, and flowers were macerated in methanol for 10 days at
room temperature. Extraction was repeated at least five times,
and the filtrates of all portions were combined. The solvent
was removed by evaporation under vacuum on reduced
pressure at 4045 C using a rotary evaporator. Water residues
were removed by lyophilization and packed in a glass pot to
store in a refrigerator.
2.3. Fractions Preparation. The dry methanolic crude extracts
(20 g) were resuspended in a methanol : water solution (1 : 1)
and extracted with hexane (3 500 mL). After methanol
removal by evaporation, the residual aqueous solution was
subjected to a sequential partition with dichloromethane (3
500 mL) and ethyl acetate (3 500 mL). The solvent of each
fraction, including the final aqueous fraction, was removed as
described above.
2.4. Antimicrobial Activity
2.4.1. Microorganisms. The antimicrobial effect of various
extract samples was assayed against Staphylococcus aureus
CCMB262 (streptomycin and dihydrostreptomycin resistant), Staphylococcus aureus CCMB263 (novobiocin resistant), Bacillus cereus CCMB 282, Escherichia coli CCMB261
(sulfonamide resistant), Pseudomonas aeruginosa CCMB268,
Candida albicans CCMB266 (fluconazole and amphotericin
B resistant), and Candida parapsilosis CCMB 288 (fluconazole and amphotericin B resistant) from the Culture Collection of Microorganisms of Bahia (CCMB).

Evidence-Based Complementary and Alternative Medicine

2.4.2. Agar Disk Diffusion Method (ADD). A screening for
antimicrobial activity was performed by Agar Disk Diffusion
method, according to the Clinical and Laboratory Standards
Institute [12], with some modifications. Methanolic solutions
of the extracts at 200 mgmL1 were sterilized by filtration
through a 0.22 m membrane filter. Filter paper disks (
6 mm) were impregnated with 5 L of each extract solution
(1 mg of crude extract/disk), and the methanol was evaporated at room temperature for 2 h. The microorganisms
were grown on Agar Type 1 Himedia-RM 666 and Mueller
Hinton Himedia-M391 broth. After 18 h (bacteria) and 36 h
(yeast), the inoculum was adjusted to 5 105 UFCmL1
and 1.5 108 UFCmL1 , respectively, in saline solution at
0.45%. Microorganism suspensions (100 L) were uniformly
spread in Petri plates containing Mueller-Hinton Agar. The
paper disks impregnated with the samples were placed on the
surface of the agar. The plates were incubated at 28 C/48 h and
37 C/18 24 h for yeasts and bacteria, respectively. Inhibition
zones (mm) were measured, and a positive result was considered with halos larger than 6.5 mm (disk diameter included).
Nystatin (10 g/disk), erythromycin, and chloramphenicol
(30 g/disk) were used as positive controls against yeasts and
bacteria. The tests were done in triplicate.
2.4.3. Minimum Inhibitory Concentration (MIC) Tests. The
determination of the Minimum Inhibitory Concentration
was performed as described in the CLSI [13, 14], with
modifications. The extracts or fractions (42 mg) solubilized in DMSO 50% were sterilized by filtration through
a 0.22 m membrane filter. In 96-well plates, 95 L of
extracts solution and 95 L of Mueller-Hinton broth (2X
concentrated) were conditioned in the first well, and the
seriated dilutions were carried out in all subsequent wells.
The range of evaluated extract concentration was from
21.05 mgmL1 to 0.01 mgmL1 . Cultures of 18 h (bacteria)
and 36 h (yeast) were collected to saline solution 0.45%, and
5 L of microorganism suspension at 9 106 UFCmL1 and
5 105 UFCmL1 (bacteria and yeasts, resp.) was added
in each well. The microplates were incubated at 28 C/48 h
for yeasts and 37 C/24 h for bacteria. An aqueous solution
of 2,3,5-triphenyltetrazolium chloride (50 L at 5 mgmL1 )
for yeasts or rezasurin (30 L at 0,1 mgmL1 ) for bacteria
was added in all wells. The result was read after 3 h of
incubation, and red wells were considered an indication
of microbial growth. The MIC was defined as the lowest
concentration in which there was no visible growth after
incubation. Controls with nystatin for yeasts (10 mgmL1 )
and chloramphenicol (20 mgmL1 ) for bacteria were made.
Controls of the microbial strains viability, sample sterility, and
water were also performed. The MIC of the DMSO solution
in water (50%) was also determined. All tests were done
in triplicate. Plant extracts with MICs 2.631 mgmL1 were
considered active antimicrobial agents.
2.5. Phytochemical Screening. A phytochemical screening
was performed on all fractions of L. origanoides by thin layer
chromatography, according to the procedure described in the
TLC Atlas Plant Drug Analysis [15].

Evidence-Based Complementary and Alternative Medicine

Table 1: Antimicrobial activity by disk diffusion method of crude extracts from L. origanoides, L. alnifolia, L. thymoides, and reference
antibiotics.
E. coli
CCMB261

S. aureus
CCMB262

Inhibition zones (mm)1

S. aureus
P. aeruginosa
CCMB263
CCMB268

L. alnifolia leaves
L. alnifolia stems
L. alnifolia flowers

R
R
R

10.2 1.3
12.3 1.0
8.4 0.9

10 0.5
13.9 0.8
R

L. origanoides leaves
L. origanoides stems

R
R

13.0 0.5
R

L. thymoides leaves
L. thymoides stems
L. thymoides flowers

R
R
R

Samples

Cloramphenicol2

C. albicans
CCMB286

C. parapsilosis
CCMB288

R
R
R

R
R
R

R
R
6.9 0.1

12.0 0.9
R

R
R

11.7 0.4
R

7.1 0.4
R

11.2 1.0
8.7 0.6
8.5 1.5

9.7 1.3
R
R

R
R
R

R
R
R

R
R
R

15.8 1.7

22.6 2.2

8.5 0.9

17.5 0.9

16.6 0.9

Nystatin3
1

Inhibition zones including the diameter disk (6 mm); 2 cloramphenicol at 30 g/disk; 3 nystatin at 10 g/disk; R: resistant; (): Not evaluated.

Table 2: Minimum inhibitory concentration (MIC) of the crude extract and fractions from L. origanoides and reference antibiotics.
Samples

E. coli
CCMB261

CEXL
HEFL
DIFL
EAFL
AQFL

5.263
1.316
0.658
0.658
2.631

CEXS
HEFS
DIFS
EAFS
AQFS

5.263
2.631
0.658
1.316
5.263

Minimum inhibitory concentration (mgmL1 )

S. aureus
S. aureus
P. aeruginosa
C. albicans
CCMB262
CCMB263
CCMB268
CCMB286
L. origanoides leaves
1.316
0.658
5.263
2.631

0.329

2.631

2.631

1.316

0.329

2.631

2.631

2.631
L. origanoides stems
5.263
2.631
5.263
2.631

0.658

2.631

2.631

0.658

2.631

2.631

5.263

2.631

C. parapsilosis
CCMB288
2.631

5.263

Chloramphenicol

10

0.078

0.313

Nystatin

1.25

2.5

(): not evaluated; CEX: crude extract; HEF: hexane fraction; DIF: dichloromethane fraction; EAF: ethyl acetate fraction; AQF: aqueous fraction; L: leaves; S:
stems. MIC = 2.631 mgmL1 was the maximum value considered for attribution of antimicrobial activity for each strain.

3. Results and Discussion

The disk diffusion assay results are summarized in Table 1.
All extracts have shown antimicrobial activity, inhibiting
at least one of the evaluated microorganisms, as for the
flowers and stems extracts of L. thymoides. A wide spectrum
antimicrobial activity was noted with L. origanoides leaves,
inhibiting the growth of two S. aureus strains, in addition to
C. albicans and C. parapsilosis.
S. aureus CCMB262 was the most sensitive strain concerning theantibacterial effect of the extracts (seven out of
eight extracts were active), whereas E. coli and P. aeruginosa
were the least sensitive microorganisms (all extracts inactive).
In general, the Gram-positive bacteria were the most sensitive
to the tested methanolic extracts, with inhibition zones of 6.9
to 12.3 mm. This higher resistance of Gram-negative bacteria

was expected, due to the polysaccharide outer membrane

with a lower permeability and efflux pumps. This system
prevents the accumulation of the antimicrobial agent inside
the cell, thus impeding the substance from reaching its target
or the concentration becoming lethal to the cell [16].
Despite the low permeability of the fungal cell walls,
composed of 1,3--glucan and chitin polymers [17], an
anticandidal activity was observed in L. alnifolia flowers,
as well as in L. origanoides leaves. C. parapsilosis was the
most sensitive yeast, which was inhibited by two out of eight
extracts.
L. origanoides was selected for further assays (fractionation by liquid-liquid partition, MIC determination, and
phytochemical screening) because of its wide spectrum
of antimicrobial activity. The MIC determination (Table 2)

Evidence-Based Complementary and Alternative Medicine

Table 3: Phytochemical screening results of the fractions of L. origanoides stems and leaves.

Reagent
Metabolites

AS
Terpenes and
steroids

DRG
Alkaloids

LB
Triterpenes and
steroids
L. origanoides leaves
+

KOH
Coumarins and
anthraquinones

NP/PEG
Flavonoids and
phenolic acids

Saponins

HEFL

++

DIFL

EAFL

++

AQFL

HEFS

++

+
L. origanoides stems
+

DIFS

EAFS

++

AQFS

AS: anisaldehyde sulfuric acid reagent; DRG: Dragendorff reagent; LB: Liebermann-Burchard reagent; KOH: potassium hydroxide reagent; NP/PEG: natural
products/polyethylene glycol reagent; CEX: crude extract; HEF: hexane fraction; DIF: dichloromethane fraction; EAF: ethyl acetate fraction; AQF: aqueous
fraction; L: leaves; S: stems; (): not detectable; (+): present; (++): highly present.

showed that leaf crude extracts also inhibited the microbial

growth of all evaluated Gram-positive bacteria and yeasts
(MIC ranging from 2.631 to 0.658 mgmL1 ), in agreement
with the antimicrobial results by the previous ADD test.
However, stem methanol extracts, although inactive in the
ADD assay, showed action against S. aureus CCMB263 and
C. albicans, in the latter with MIC values similar to those
obtained by the leaf extracts. The reason for the difference
in sensitivity between the two methods might be related to
diffusion properties of the antimicrobial compounds from
the extracts into the agar and adsorption in the paper disk
[18].
The fractions from leaves and stems exhibited varying
degrees of antimicrobial action, with hexane and ethyl
acetate fractions from leaves showing the highest antibacterial effect against S. aureus (MIC0.329 mgmL1 ). An
enhanced activity against all microorganisms was observed
with the fractionation, including against E. coli, to which the
extracts were considered inactive. Dichloromethane fractions
from leaves and stems, as well as the ethyl acetate fraction
from leaves, were the most efficient against E. coli (MIC
0.658 mgmL1 ). The best result against C. albicans was
obtained by the dichloromethane fraction from the stems
(MIC0.658 mgmL1 ), a noteworthy result considering that
invasive candidiasis is a leading cause of mycosis-mortality
[19].
The results of phytochemical screening of L. origanoides fractions showed the presence of terpenes, steroids,
coumarins, saponins, flavonoids, and phenolic acids in stems
and leaves, while alkaloids were absent (Table 3). These
phytoconstituents, common in Lippia [5], are recognized
as antimicrobial agents against several bacteria and yeasts
[20]. Terpenes were the predominant metabolites in the
hexane fractions and flavonoids in ethyl acetate fractions.
These constituents may be the responsible for the improved
antibacterial activity in hexane and ethyl acetate fractions.

4. Conclusions
The antimicrobial activity by ADD gives preliminary scientific evidence to the traditional use of L. alnifolia, L.
origanoides, and L. thymoides as antiseptic agents and in
the treatment of infectious diseases. Although the results
of this study suggest the antibacterial potential of three
evaluated species, the L. origanoides leaves exhibited the
widest antimicrobial action being a promising alternative
in the treatment of bacterial and fungal infections. As the
fractionation of L. origanoides crude extracts improved the
activity in spectrum and intensity, further investigations are
in progress to isolate and characterize the active metabolites.

Conflict of Interests
The authors have declared that they have no conflict of
interests.

Acknowledgments
The authors thank the Instituto do Milenio do Semiarido
and Conselho Nacional de Desenvolvimento Cientfico e
Tecnologico (IMSEAR/CNPq) for the financial support,
Fundacao de Amparo a Pesquisa do Estado da Bahia
(FAPESB) for the financial support, Universidade Estadual
de Feira de Santana for the fellowship granted to Velize D.
Rodrigues, and CNPQ for the fellowship granted to Tania
Regina dos Santos Silva.

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