Antimicrobial Activity of Gentiana lutea L. Extracts

Abstract

Methanolic extracts of flowers and leaves of Gentiana lutea L., together with the isolated compounds mangiferin, isogentisin and gentiopicrin, were used to investigate the antimicrobial activity of the plant. A variety of Gram-positive and Gram-negative bacteria as well as the yeast Candida albicans has been included in this study. Both extracts and isolated compounds showed antimicrobial activity with MIC values ranging from 0.12-0.31 mg/ml. Our study indicated that the synergistic activity of the pure compounds may be responsible for the good antimicrobial effect of the extracts. Quantification of the secondary metabolites was performed using HPLC.

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Introduction
The roots of Gentiana lutea L. (Gentianaceae),
a yellow fl owering plant commonly found in the
mountain regions of central and south Europe,
are very popular as a stomachic as well as a com-
ponent in preparations showing benefi cial effects
in gall and liver diseases (Wichtl, 1994). The ac-
tive principles are the bitter tasting secoiridoid
glycosides gentiopicrin and amarogentin. Some
investigations pointed out an interesting chemi-
cal composition of the aerial parts of G. lutea.
The presence of the xanthone isogentisin and two
fl avone heterosides was reported in leaves of G.
lutea (Hostettmann et al., 1973). The secoiridoids
gentiopicrin and swertiamarin, the xanthones
mangiferin, isogentisin and isogentisin-3-O-pri-
meveroside, and the fl avones isoorientin and iso-
vitexin have been isolated from the aerial parts of
G. lutea (Menkovic´ et al., 2000).
The development of resistance by pathogens to
many of the commonly used antibiotics provides
a stimulus for further attempts to search for new
antimicrobial agents to combat infections and
overcome problems of resistance and side effects
of the currently available antimicrobial agents.
Antibacterial effects of G. lutea roots have been
described recently, and it was shown that the
dry extract was effective against Streptococcus
pyogenes (Weckesser et al., 2007). Other studies
indicated that G. lutea exhibits antimicrobial ef-
fects that correspond to the effect of ampicillin
and it could be used in the treatment of bacterial
infections (Stierna et al., 2005). As for the antimi-
crobial activity of leaves and fl owers extracts is
concerned, only an antitubercular effect against
Mycobacterium bovis was reported (Menkovic´ et
al., 1999). The aim of the present study was to
investigate the antimicrobial activity of G. lutea
leaves and fl owers extracts and the isolated com-
pounds gentiopicrin, isogentisin and mangiferin
against various bacteria and the yeast Candida
albicans.
Material and Methods
Plant material
Leaves and fl owers of Gentiana lutea were col-
lected at mountain Suvobor (at a height of ca.
830 m), Serbia, in July 2006. A voucher specimen
(17506) has been deposited in the herbarium of
the Botanic Garden “Jevremovac”, Faculty of Bi-
ology, University of Belgrade, Serbia.
Sample preparation
Air-dried leaves and fl owers were extracted
separately with methanol (1000 ml) in a Soxhlet
apparatus for 24 h and the solvent was evapo-
rated. Dry extracts of leaves (17.5 g) and fl owers
(22.3 g) were used for the experiments.
Antimicrobial Activity of Gentiana lutea L. Extracts
Katarina Šavikin*, Nebojša Menkovic´, Gordana Zdunic´, Tatjana Stevic´,
Dragoja Radanovic´, and Teodora Jankovic´
Institute for Medicinal Plants Research, Tadeuša Košc´uška 1, 11000 Belgrade, Serbia.
Fax: +38 11 13 03 16 49. E-mail: ksavikin@mocbilja.rs
* Author for correspondence and reprint requests
Z. Naturforsch. 64 c, 339 – 342 (2009); received November 14/December 26, 2008
Methanolic extracts of fl owers and leaves of Gentiana lutea L., together with the isolated
compounds mangiferin, isogentisin and gentiopicrin, were used to investigate the antimicro-
bial activity of the plant. A variety of Gram-positive and Gram-negative bacteria as well
as the yeast Candida albicans has been included in this study. Both extracts and isolated
compounds showed antimicrobial activity with MIC values ranging from 0.12 – 0.31 mg/ml.
Our study indicated that the synergistic activity of the pure compounds may be responsible
for the good antimicrobial effect of the extracts. Quantifi cation of the secondary metabolites
was performed using HPLC.
Key words: Gentiana lutea, Antimicrobial Activity, Gentiopicrin
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340 K. Šavikin et al. · Antimicrobial Activity of Gentiana lutea
HPLC conditions
Analyses were carried out on a HP series
1090 instrument with a DAD detector, on a re-
verse phase Zorbax SB-C18 analytical column
[150 × 4.6 mm i.d., particle size 5 μm (Agilent)].
Mobile phase A was H
2
O containing 1% 0.1
N
H
3
PO
4
, mobile phase B was MeCN. Gradient
elution was according to the following scheme:
98 – 90% A, 0 – 5 min; 90% A, 5 – 10 min; 90 – 85%
A, 10 – 13 min; 85% A, 13 – 15 min; 85 – 70% A,
15 – 20 min; 70 – 40% A, 20 – 24 min; 40 – 0% A,
24 – 28 min; fl ow at 1 ml/min; detection at 260 and
320 nm. The xanthones mangiferin and isogenti-
sin, and the secoiridoid gentiopicrin were isolated
according to the previously published procedure
(Menkovic´ et al., 2000). Quantifi cation was per-
formed using HPLC and the amounts of the com-
pounds were calculated using calibration curves.
All experiments were repeated at least three
times. The results are presented as mg/g of dry
weight (dw).
Studied activity
The antimicrobial activity was tested against
six Gram-negative (Escherichia coli, Salmonella
typhimurium, S. enteritidis, Pseudomonas aerugi-
nosa, P. tolaasii, Enterobacter cloacae) and nine
Gram-positive bacteria (Staphylococcus aureus, S.
epidermidis, Streptococcus faecalis, Bacillus subti-
lis, Micrococcus luteus, M. fl avus, Proteus mirabi-
lis, Sarcina lutea, Listeria monocytogenes), as well
as one human pathogen yeast (Candida albicans).
The MIC (minimum inhibitory concentration)
values were determined using the broth microdi-
lution method in 96-hole plates according to NC-
CLS (2000). Serial dilutions of the stock solutions
of test extracts in broth medium (Muller-Hinton
broth or Sabouraud broth) were prepared in a
microtiter plate. The microbial suspensions were
added in the microwells at the concentration of
5 · 10
5
organisms/ml. The MIC values were de-
termined as the lowest concentrations preventing
visible growth. Streptomycin and nystatin were
used as a positive control. Each assay was repeat-
ed independently two times.
Results and Discussion
The HPLC profi les of G. lutea leaves and fl ow-
ers methanolic extracts are shown in Fig. 1. The
amounts of mangiferin and gentiopicrin were
nearly similar in leaves and fl owers, but the
amount of isogentisin was about ten times higher
in fl owers (Table I).
The results of the antimicrobial activity deter-
mination of G. lutea extracts and isolated com-
pounds are presented in Table II. Leaves and
fl owers extracts inhibited the growth of 15 of
16 pathogenic microorganisms tested, only the
Gram-positive bacterium Listeria monocytogenes
was resistant and has grown at the highest ap-
plied concentrations of both extracts. The MIC
values of the leaves extract were between 0.12
and 0.31 mg/ml, and the most sensitive to this ex-
tract were Pseudomonas aeruginosa, Bacillus sub-
tilis, Proteus mirabilis, Staphylococcus epidermidis
and Candida albicans. The fl owers extract exerted
slightly lower antimicrobial activity and the most
susceptible microorganism was Salmonella enteri-
tidis (MIC 0.15 mg/ml).
Among the individual extract components, the
compound with the widest spectrum of activity
was found to be gentiopicrin. It was most active
against Escherichia coli (0.12 mg/ml) and showed
moderate activity against Salmonella typhimu-
rium and Staphylococcus aureus (0.15 mg/ml).
The obtained results, along with published data,
characterize gentiopicrin as a natural compound
with a broad antimicrobial effect (Kumarasamy et
al., 2003; Nadinic et al., 2002). The antimicrobial
activity of the xanthone isogentisin against My-
cobacterium bovis has been reported previously
(Menkovic´ et al., 1999). In the present study, iso-
gentisin showed moderate antimicrobial activi-
ties with MIC values between 0.15 and 0.31 mg/
ml. Among the Gram-negative bacteria the most
susceptible were E. coli and Pseudomonas aeru-
ginosa. The species most sensitive to isogentisin
among the Gram-positive bacteria was Micro-
coccus luteus. Compared to the other examined
compounds, mangiferin showed lower antibacte-
rial activity with MIC values between 0.20 and
0.31 mg/ml. The antimicrobial activity of mangif-
Table I. The amount of secondary metabolites in G.
lutea leaves and fl owers extracts.
Sample Gentiopicrin
a
[mg/g dw] Mangiferin
a
[mg/g dw] Isogentisin
a
[mg/g dw]
Leaves 38.85 ± 0.7 9.57 ± 0.4 12.86 ± 0.7
Flowers 48.38 ± 1.4 8.98 ± 0.4 123.23 ± 3.1
a
Mean ± s.d. (n = 3).
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K. Šavikin et al. · Antimicrobial Activity of Gentiana lutea 341
Table II. Antimicrobial activity (MIC values in mg/ml) of G. lutea leaves and fl owers extracts and isolated com-
pounds
a
.
Microorganism Extract 1 Extract 2 MG IG GP Streptomycin
(nystatin*)
Bacillus subtilis ATCC 6051 0.12 0.19 0.27 0.19 0.19 0.0052
Listeria monocytogenes ATCC 15313 0.31 0.31 0.31 0.22 0.27 0.016
Micrococcus fl avus ATCC 10786 0.31 0.22 0.22 0.22 0.27 0.0052
Micrococcus luteus ATCC 10240 0.15 0.19 0.20 0.15 0.31 0.016
Proteus mirabilis ATCC 14273 0.12 0.19 0.20 0.19 0.19 0.0052
Sarcina lutea ATCC 10054 0.27 0.19 0.31 0.31 0.22 0.038
Staphylococcus aureus ATCC 25932 0.15 0.22 0.31 0.22 0.15 0.0052
Staphylococcus epidermidis ATCC 12228 0.12 0.19 0.27 0.22 0.22 0.0052
Streptococcus faecalis ATCC 12952 0.27 0.22 0.22 0.19 0.19 0.027
Escherichia coli ATCC 25922 0.15 0.19 0.20 0.15 0.12 0.0052
Enterobacter cloacae ATCC 13883 0.22 0.27 0.27 0.31 0.19 0.038
Pseudomonas aeruginosa ATCC 27853 0.12 0.27 0.27 0.15 0.22 0.016
Pseudomonas tolaasii NCTC 387 0.27 0.27 0.27 0.31 0.22 0.027
Salmonella typhimurium ATCC 14028 0.15 0.19 0.22 0.19 0.15 0.038
Salmonella enteritidis ATCC 13076 0.19 0.15 0.22 0.19 0.19 0.016
Candida albicans ATCC 10231 0.12 0.22 0.31 0.27 0.27 0.0052*
a
Extract 1, methanolic extract of G. lutea leaves; extract 2, methanolic extract of G. lutea fl owers; MG, mangiferin;
IG, isogentisin; GP, gentiopicrin.
min
5 10 15 20 25 30
mAU
0
50
100
150
200
250
Mangiferin
Gentiopicrin
Isogentisin
G. lutea, leaves
min
5 10 15 20 25 30
mAU
0
200
400
600
800
1000
1200
1400
G. lutea, flowers
Mangiferin
Isogentisin
Gentiopicrin
Fig. 1. HPLC chromatograms of methanolic extracts of G. lutea leaves and fl owers.
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342 K. Šavikin et al. · Antimicrobial Activity of Gentiana lutea
erin against several bacterial species has been re-
ported previously (Stoilova et al., 2005).
Our study indicated that each tested compound
did not possess a dominant role in the antimicro-
bial activity of crude extracts. Thus, synergistic ac-
tivity may be responsible for the inhibitory effect
of the extracts. Since crude extracts of G. lutea
leaves and fl owers showed a wide range of an-
timicrobial effect, their use in the treatment of
various bacterial and fungal infections could be
benefi cial.
Acknowledgements
The authors acknowledge their gratitude to the
Ministry of Science of Serbia for fi nancial support
(project number TR 6846B).
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