Antibacterial and antioxidant activities of ursolic acid and derivatives

Article abstract of DOI:10.3390/molecules19011317

Ursolic acid, an important bioactive compound, was isolated from ethanol extract of aerial parts of Sambucus australis. In order to develop bioactive ursolic acid derivatives, two semi-synthetic compounds were obtained through modification at C-3. The antibacterial activity of the ursolic acid and its derivatives was investigated. The microdilution method was used for determination of the minimal inhibitory concentration (MIC), against twelve bacterial strains. The influence of ursolic acid and its derivatives on the susceptibility of some bacterial pathogens to the aminoglycosides antibiotics neomycin, amikacin, kanamycin and gentamicin was evaluated. The most representative synergistic effect was observed by 3ss-formyloxy-urs-12-en- 28-oic acid at the concentration of 64 g/mL in combination with kanamycin against Escherichia coli (27), a multidrugresistant clinical isolate from sputum, with reduction of MIC value from 128 g/mL to 8 g/mL. Ursolic acid and its derivatives were examined for their radical scavenger activity using the DPPH assay, and showed significant activity.

Full text of DOI:10.3390/molecules19011317

Molecules 2014, 19, 1317-1327; doi:10.3390/molecules19011317
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
Antibacterial and Antioxidant Activities of Ursolic Acid
and Derivatives
1
1,
1
Patrícia G.G. do Nascimento , Telma L.G. Lemos *, Ayla M.C. Bizerra ,
1
1
2
Ângela M.C. Arriaga , Daniele A. Ferreira , Gilvandete M.P. Santiago ,
3
4
Raimundo Braz-Filho and José Galberto M. Costa
1
Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará,
Fortaleza-CE 60451-970, Brazil; E-Mails: georgina.quimica@gmail.com (P.G.G.N.);
aylamarcia@yahoo.com.br (A.M.C.B.); angelamcarriaga@yahoo.com.br (A.M.C.A.);
dafufc@yahoo.com.br (D.A.F.)
2
3
4
Departamento de Farmácia, Universidade Federal do Ceará, Fortaleza-CE 60430-370, Brazil;
E-Mail: gil@ufc.br
Laboratório de Ciências Químicas (LCQUI)-CCT-UENF/PPGQO-DEQUIM-UFRRJ,
Campos dos Goytacazes-RJ 28013-602, Brazil; E-Mail: braz@uenf.br
Laboratório de Pesquisa de Produtos Naturais, Universidade Regional do Cariri,
Crato-CE 63105-000, Brazil; E-Mail: galberto.martins@gmail.com
*
Author to whom correspondence should be addressed; E-Mail: tlglemos@yahoo.com.br;
Tel.: +55-85-3366-9366; Fax: +55-85-3366-9978.
Received: 7 November 2013; in revised form: 11 December 2013 / Accepted: 20 December 2013 /
Published: 21 January 2014
Abstract: Ursolic acid, an important bioactive compound, was isolated from ethanol
extract of aerial parts of Sambucus australis. In order to develop bioactive ursolic acid
derivatives, two semi-synthetic compounds were obtained through modification at C-3.
The antibacterial activity of the ursolic acid and its derivatives was investigated. The
microdilution method was used for determination of the minimal inhibitory concentration
(
MIC), against twelve bacterial strains. The influence of ursolic acid and its derivatives on
the susceptibility of some bacterial pathogens to the aminoglycosides antibiotics neomycin,
amikacin, kanamycin and gentamicin was evaluated. The most representative synergistic
effect was observed by 3β-formyloxy-urs-12-en-28-oic acid at the concentration of
6
4 μg/mL in combination with kanamycin against Escherichia coli (27), a multidrug-
resistant clinical isolate from sputum, with reduction of MIC value from 128 μg/mL to

Molecules 2014, 19
1318
8
μg/mL. Ursolic acid and its derivatives were examined for their radical scavenger activity
using the DPPH assay, and showed significant activity.
Keywords: Sambucus australis; ursolic acid; antioxidant activity; antibacterial activity
1
. Introduction
Ursolic acid (1), an ursane-type pentacyclic triterpene, is a constituent of certain medicinal herbs
and is also found in fruits [1]. This triterpenoid is the major secondary metabolite isolated from the
ethanol extract of aerial parts of Sambucus australis Cham. & Schltdl (Caprifoliaceae), a shrub,
popularly known as “sabugueiro”.
It is well known to possess a wide range of biological activities including anti-inflammatory [2],
anticancer [3], hypoglycemic [4], antiprotozoal against Plasmodium falciparum [5], antioxidant [1,6,7],
antibacterial [8], and prevents abdominal adiposity [9]. The antibacterial properties of pentacyclic
triterpenes and their derivatives have been extensively studied [10–13], and the activity of these
compounds also resides in their potential to enhance bacterial susceptibility to other compounds,
including antibiotics [14].
Antibiotic resistance is a serious problem in the area of public health. The search for new
therapeutic agents that can help patients infected by bacterial agents is a challenge for all professionals
in the field. The use of natural products is an alternative that can produce good results.
The production of oxygen and free radicals in the body, probably involves the development of
many diseases such as inflammation, cancer, rheumatoid arthritis, Parkinson's and Alzheimer's
diseases [15]. The natural antioxidants cause less toxic side effect and can provide protection against
oxidative degradation by decrease free radicals in cosmetic, pharmaceutical, and food.
The aim of the present study was to prepare derivatives of ursolic acid (1), and to investigate the
antibacterial and scavenger activities using DPPH assay of one 1 and its derivatives 1a and 1b. The
influence of the compounds 1, 1a and 1b on the susceptibility of several Gram-positive and Gram-negative
bacteria towards the aminoglycoside antibiotics neomycin, amikacin, kanamycin and gentamicin was
also evaluated.
2
. Results and Discussion
2.1. Synthesis
Ethanol extract of the aerial parts of S. australis (SAEtOH) was fractionated and purified by
classical chromatographic methods to isolate the triterpenoid ursolic acid (1) [16]. In order to develop
bioactive ursolic acid derivatives, two semi-synthetic compounds were obtained through modification
at C-3. The structural changes involving the hydroxyl group were acetylation and formylation (Scheme 1).
1
13
The structures of all compounds were established by 1D H and C (and DEPT) and 2D HSQC and
HMBC NMR spectral data and by comparing their spectroscopy data with the literature [16–18].
The treatment of (1) with acetic anhydride and pyridine at room temperature afforded 3β-acetoxy-
1
urs-12-en-28-oic acid (1a) in quantitative yield. The H-NMR spectrum of 1a showed a signal at δ
H

Molecules 2014, 19
1319
2
.05 typical of hydrogen of acetyl group. The presence of this group was further confirmed by the
13
1
appearance of the signal at δ 170.9 in the C- NMR spectrum [17]. Analysis of the H-NMR
C
H
spectrum of 3β-formiloxy-urs-12-en-28-oic acid (1b) showed a signal at δ 8.12 for the formyl group
confirmed by the presence of a signal at δ 161.3 attributed to a carbonyl [18].
C
Scheme 1. Synthesis of ursolic acid derivatives.
29
30
2
0
7
2
1
2
1
9
1
2
18
2
1
11
25
26 13
14
C28OOH
1
9
1
6
a
2
O
1
0
8
15
A
4
A
3
3
7
27
5
O
HO
6
b
1a
24
23
1
O
A
3
H
O
1
c
2 2
Reagents and Conditions: (a) Ac O, pyridine/rt/24 h; (b) HCO H, perchloric acid/60 °C/4 h.
2
.2. Antimicrobial Activity and Evaluation of the Modulatory Activity by Direct Contact
Ursolic acid (1), 3β-acetoxy-urs-12-en-28-oic acid (1a) and 3β-formyloxy-urs-12-en-28-oic acid
(
1b) were tested for antibacterial activity against twelve bacterial strains (different strains of
Staphylococcus aureus, Bacillus cereus, two strains of Escherichia coli, Pseudomonas aeruginosa,
Aeromonas caveae, Klebsiella pneumoniae, Shigella flexneri, Vibrio colareae and Listeria
monocytogenes), by employing the microdilution method. The corresponding minimum inhibitory
concentration (MIC) values of (1), (1a) and (1b) are shown in Table 1.
Although the antibacterial activity of ursolic acid (1) against several bacterial strains has been
reported in the literature [8,10–12], in our case compound 1 showed activity against six bacterial
strains, and the best result was found against S. aureus (ATCC 6538), with a MIC value of 32 μg/mL.
Ursolic acid (1) was also effective against E. coli (ATCC 25922), K. pneumoniae and S. flexneri with a
MIC value of 64 μg/mL in the three cases.
3
β-Acetoxy-urs-12-en-28-oic acid (1a) showed significant activity (in terms of clinical MIC ≤
1
,024 μg/mL) against all tested strains except for S. aureus (ATCC 12624). The best results were
found against E. coli (ATCC 25922) and S. flexneri with a MIC value of 32 μg/mL in both cases,
K. pneumoniae and S. aureus (ATCC 6538) with MIC values of 64 μg/mL and 128 μg/mL,
respectively (Table 1). The literature reports activity against Streptococcus pneumoniae (ATCC 6305)
for compound (1a) [11], however, this is the first report on the antibacterial activity of the derivative
1
a on the bacterial strains used.
In the present study, 3β-formyloxy-urs-12-en-28-oic acid (1b) presented significant activity from a
clinical point of view against ten evaluated bacterial strains, with emphasis against S. aureus (ATCC

Molecules 2014, 19
1320
6
538), A. caveae and L. monocytogenes with MIC values of 256 μg/mL, and K pneumoniae and
S. flexneri with MIC values of 128 μg/mL (Table 1).
Table 1. Values of the minimal inhibitory concentration (MIC) of compounds 1, 1a and 1b.
MIC (µg/mL)
Bacterial strains
1
1a
256
≥1024
128
512
32
1b
512
Staphylococcus aureus (ATCC 12692)
Staphylococcus aureus (ATCC 12624)
Staphylococcus aureus (ATCC 6538)
Bacillus cereus (ATCC 33018)
≥1024
≥1024
32
≥1024
256
≥1024
64
512
Escherichia coli (ATCC 25922)
512
Escherichia coli (ATCC 27)
512
256
512
256
64
512
Pseudomonas aeruginosa (ATCC 15442)
Aeromonas caveae (ATCC 15468)
Klebsiella pneumoniae (ATCC 10031)
Shigella flexneri (ATCC 12022)
Vibrio colareae (ATCC 15748)
512
512
≥1024
64
256
128
64
32
128
≥1024
≥1024
512
256
≥1024
256
Listeria monocytogenes (ATCC 19117)
(
1) ursolic acid, (1a) 3β-acetoxy-urs-12-en-28-oic acid, (1b) 3β-formyloxy-urs-12-en-28-oic acid.
Interestingly, among all evaluated bacterial strains in this study, S. aureus (ATCC 12624) was the
most resistant to the tested compounds 1, 1a and 1b.
The effects on the MIC values of the association of compounds 1, 1a and 1b with aminoglycoside
antibiotics are shown in Table 2. In these assays the compounds that showed MIC ≤ 1024 μg/mL in the
MIC determination were used.
Table 2 indicate a synergism between ursolic acid (1) and the aminoglycoside antibiotics in almost
all microorganisms, except for gentamicin and amikacin against E. coli (ATCC 25922), and
kanamycin against P. aeruginosa and K. pneumoniae. Ursolic acid (1) was found to enhance the
susceptibility of Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes to
the β-lactam antibiotics ampicilin and oxacilin [14], but there is no report in the literature about this
compound acting synergistically with the aminoglycoside antibiotics neomycin, amikacin, kanamycin
and gentamicin.
The association of 3β-acetoxy-urs-12-en-28-oic acid (1a) with the aminoglycoside antibiotics
increased the effect of these antibiotics against the strains evaluated; thus 1a may be considered an
excellent enhancer of the mechanism of action of these aminoglycoside antibiotics.
The association of 3β-formyloxy-urs-12-en-28-oic acid (1b) with the aminoglycoside antibiotics
showed synergistic effects against several microorganisms. It is also possible to demonstrate that there
was no potential interference on the activity of gentamicin against B. cereus and S. aureus (ATCC
1
2692) when associated to 3β-formyloxy-urs-12-en-28-oic acid (1b) and the associations with
amikacin against K. pneumoniae and kanamycin against L. monocytogenes.

Molecules 2014, 19
Table 2. Combinating testing of ursolic acid (1), 3β-acetoxy-urs-12-en-28-oic acid (1a)
1321
and 3β-formiloxy-urs-12-en-28-oic acid (1b) plus neomycin, amikacin, kanamycin and
gentamicin against bacterial strains.
Combination tested
MIC (µg/mL)
Bacterial strains
Antibiotic + Substance (µg/mL) Neomycin Amikacin Kanamycin Gentamicin
S. aureus 12692
*
128
16
64
4
64
8
128
32
128
64
4
1
a (32)
b (64)
*
1
64
32
128
32
32
64
128
32
64
64
64
32
32
128
32
32
16
128
32
32
32
128
16
64
64
32
32
64
128
32
64
32
128
32
64
32
4
16
64
16
16
32
64
8
S. aureus 6538
128
18
1
(4)
1
a (16)
b (32)
*
16
16
16
64
16
64
64
64
16
32
64
32
8
1
32
B. cereus 33018
E. coli 25922
128
64
1
a (64)
b (64)
*
1
64
32
128
64
16
64
128
16
32
8
128
32
1
(8)
1
a (4)
b (64)
*
32
1
64
E. coli 27
64
1
(8)
4
1
a (32)
b (64)
*
16
1
32
8
P. aeruginosa 15442
128
64
64
64
16
16
128
32
32
64
64
16
32
64
16
16
16
64
32
64
128
16
32
16
4
1
(64)
a (64)
b (64)
*
1
1
64
32
8
A. caveae 15468
64
128
32
32
128
16
64
64
64
16
8
1
a (32)
b (64)
*
8
1
16
K. pneumonia 10031
256
128
64
1
(8)
1
a (8)
b (16)
*
1
128
512
64
S. flexneri 12022
1
(8)
1
a (4)
b (16)
*
256
128
128
16
1
32
64
8
L. monocytogenes 19117
V. colareae 15748
1
a (32)
b (32)
*
1
32
16
128
32
64
1
a (64)
16
*
only antibiotics.

Molecules 2014, 19
.3. Antioxidant Activity
Compounds 1 and 1a showed antioxidant activity by inhibiting DPPH. The compounds strongly
1322
2
−
2
−3
−2
scavenged DPPH radical, with IC values of 5.97 × 10 ± 1 × 10 and 0.73 ± 9.3 × 10 mg/mL,
50
−3
respectively. Trolox and Vitamin C, used as positive controls, showed IC values of 2.6 × 10 ±
5
0
−4
−2
−2
2
.3 × 10 and 4.3 × 10 ± 1.9 × 10 mg/mL, respectively (Table 3). Compound 1b was inactive in
the DPPH assay.
Table 3. IC values of ursolic acid (1) and derivatives (1a and 1b).
5
0
Samples
1
IC50 (mg/mL)
−2
−3
5.97 × 10 ± 1 × 10
−
2
1
a
0.73 ± 9.3 × 10
not active
1
b
−
3
−4
Trolox
2.6 × 10 ± 2.3 × 10
−
2
−2
Vitamin C
4.3 × 10 ± 1.9 × 10
3. Experimental
3.1. General
Melting points were determined on a digital Mettler Toledo FP82HT apparatus and are uncorrected.
®
The IR spectra were measured in KBr pellets using a Perkin-Elmer FT-IR Spectrum 1000. A Bruker
®
Avance DRX 500 and a Bruker Avance DPX 300 spectrometers, operating at 500 MHz and 300 MHz
1
13
for H-NMR, and 125 MHz and 75 MHz for C-NMR were used for experiments 1D and 2D with
chemical shifts given in ppm. The spectra were run using CDCl and pyridine-d as the solvent.
3
5
Chemical shifts, measured on the δ scale. The absorptions in the region of the ultraviolet (UV) visible
were obtained on a Varian Cary 50 Conc spectrophotometer. The low resolution mass spectra were
obtained by electron impact at 70 eV spectrometer with Shimadzu a QP 5000, DI-50 instrument. Silica
gel 60 (70–230 mesh) was used for column chromatography, and thin layer chromatography (TLC)
was performed on precoated silica gel G60 F254 by detection by spraying with vanillin in perchloric
acid/ethanol. All solvents used for chromatography were from Synth. The microbiological culture
media were purchased from Fundação Oswaldo Cruz—FIOCRUZ (Rio de Janeiro, Brazil).
3
.2. Plant Material
The aerial parts of Sambucus australis were collected in Guaramiranga County, State of Ceará,
northeast Brazil. A voucher specimen (#EAC15002) has been deposited at the Herbarium Prisco
Bezerra, Department of Biology, Federal University of Ceará, Brazil.
3.3. Extraction and Isolation
Aerial parts including leaves and flowers of Sambucus australis were extracted with ethyl acetate
(
1,000 g/1,500 mL) for 72 h at 25 °C, followed by ethanol (1,000 g/5,000 mL) for 72 h at 25 °C to
yield: SAEtOAc, (3.9 g, 0.39%) and SAEtOH, (1.0 g, 0.1%), respectively. The ethanol extract

Molecules 2014, 19
1323
(
SAEtOH) (1.0 g) was chromatographed on a silica gel column running a gradient of 100% ethyl
acetate to 100% methanol. The fraction eluted with 100% ethyl acetate yield a solid material, which
was further recrystallized in ethyl acetate to afforded ursolic acid (1, 180 mg, 18%); White solid; m.p.
−
1
2
79.0–281.1 °C; FTIR (cm ): 3407, 2924, 2855, 1686, 1456, 1387. MS (EI): m/z (%) 456 (0.2), 248
13
(
100), 203 (43), 133 (37), 44(83). C-NMR (C
5
D
5
N) δ ppm: 37.98 (C-1), 28.65 (C-2), 78.62 (C-3),
3
1
4
1
9.85) (C-4), 56.32 (C-5); 19.44 (C-6), 34.11 (C-7), 39.45 (C-8), 48.59 (C-9), 37.78 (C-10), 24.06 (C-11),
26.14 (C-12), 139.75 (C-13), 42.51 (C-14), 28.65 (C-15 ), 25.45 (C-16), 48.54 (C-17), 54.04 (C-18),
0.02 (C-19), 39.94 (C-20), 31.60 (C-21), 37.96 (C-22), 29.35 (C-23), 15.19 (C-24), 15.25 (C-25),
8.06 (C-26), 26.69 (C-27), 180.34 (C-28), 18.06 (C-29), 21.94 (C-30) in agreement with the literature [16].
3.4. Synthesis 3β-Acetoxy-urs-12-en-28-oic Acid (1a)
To a solution of ursolic acid (1, 50 mg, 0.109 mmol) in pyridine (0.5 mL) was added Ac
.05 mmol). After stirring at room temperature for 24 h, the reaction mixture was quenched with
(20 mL) and extracted with EtOAc 3 × 30 mL. The combined extracts were washed
O and brine, dried over anhydrous Na SO , and evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel with n-hexane/ethyl acetate = 3:7 (v:v) to
2
O (2 mL,
1
saturated CuSO
4
with H
2
2
4
−1
give a white solid; Yield 75% (40.0 mg); m.p. 175.3–178.1 °C. FTIR (cm ): 3215, 2924, 1722, 1447,
1
3
1
3
366; C-NMR (CDCl ) δ ppm: C-1 (36.94), C-2 (24.25), C-3 (81.17); C-4 (37.91); C-5 (55.51), C-6
(
(
(
(
(
18.37), C-7 (33.24), C-8 (39.71), C-9 (47.68), C-10 (37.13), C-11 (23.50), C-12 (125.93), C-13
138.18), C-14 (42.10), C-15 (28.20), C-16 (23.78), C-17 (48.18); C-18 (52.71), C-19 (39.23), C-20
39.04), C-21 (30.81), C-22 (38.47), C-23 (28.29), C-24 (17.23), C-25 (15.60), C-26 (16.91), C-27
+
23.80), C-28 (184.15), C-29 (17.33), C-30 (21.51); EIMS (m/z): 498 (0.1%, M ), 248 (17%), 43
82%) in agreement with the literature [17].
3.5. Synthesis 3β-Formiloxy-urs-12-en-28-oic Acid (1b)
2
A solution of ursolic acid (1, 50 mg, 0.109 mmol) in 89.9% HCO H (1.5 mL) and 70% perchloric
acid (6 drops) was heated in an H
allowed to cool to about 40 °C. Ac
between 55 and 60 °C until a large quantity of bubbles appeared (1 mL of Ac O was required). The
2
O bath at 60 °C for 4 h. The solution was removed from the bath and
2
O was then added dropwise while the temperature was maintained
2
2
solution was then cooled to room temperature and poured into 10 ml of H O, with stirring [19]. The
precipitate was filtered under vacuum, washed with H
2
O, and dried to give a white solid; Yield 28%
−1
13
(
23.1 mg); m.p. 117.3–119.4 °C; FTIR (cm ): 2924, 1718, 1688, 1458, 1369; C-NMR (CDCl
3
) δ
ppm: 38.53 (C-1), 23.55 (C-2), 81.35 (C-3), 37.92 (C-4), 55.57 (C-5), 18.46 (C-6), 33.11 (C-7), 39.83
(C-8), 47.75 (C-9), 37.18 (C-10), 23.97 (C-11), 125.94 (C-12), 138.27 (C-13), 42.25 (C-14), 29.94
(C-15), 24.33 (C-16), 48.22 (C-17), 52.84 (C-18), 39.10 (C-19), 39.29 (C-20), 30.86 (C-21), 36.96
(C-22), 28.27 (C-23), 21.40 (C-24), 15.75 (C-25), 16.94 (C-26), 23.84 (C-27), 183.18 (C-28), 17.25
(C-29), 17.30 (C-30) in agreement with the literature [18].

Molecules 2014, 19
.6. Antibacterial Activity and Minimal Inhibitory Concentration
The antibacterial activity of the ursolic acid and its derivatives were investigated employing a
1324
3
microdilution method, recommended by National Committee for Clinical and Laboratory Standards
M7-A6 [20]. In tests were used eight standard strains of Gram (−) and four Gram (+), and two clinical
isolates of multidrug-resistant Escherichia coli (27) (from sputum) and Staphylococcus aureus (6538)
of the surgical wound. The brain heart infusion (BHI 3.8%) broth was used for the bacterial growth (24 h,
3
5 ± 2 °C). The inoculum was an overnight culture of each bacterial species in the BHI broth diluted in
8
the same medium to a final concentration of approximately 1 × 10 CFU/mL (0.5NTU – McFarland
6
scale). After this, the suspension was dilluted to 1 × 10 CFU/mL in 10% BHI. A total of 100 μL of
5
each dilution was distributed in 96-well plates plus substance, achieving 5 × 10 CFU/mL as the final
concentration of the inoculums [21–23].
The initial solution of the ursolic acid and its derivatives were performed using 10 mg of each
extract dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain an initial concentration of
1
0 mg/mL. From this concentration, several dilutions were made in distilled water in order to obtain a
stock solution of 1,024 μg/mL. Further serial dilutions were performed by the addition of the BHI
broth to reach a final concentration in the range of 8–512 μg/mL). All experiments were performed in
triplicate and the microdilution trays were incubated at 35 ± 2 °C for 24 h. The antibacterial activity
was detected using a colorimetric method by adding 25 μL of the resauzurin staining (0.01%)
aqueous solution in each well at the end of the incubation period [24]. The minimal inhibitory
concentration (MIC) was defined as the lowest extract concentration able to inhibit the bacteria
growth, as indicated by resauzurin staining (dead bacterial cells are not able to change the staining color
by visual observation—blue to red).
3
.7. Evaluation of the Modulatory Activity by Direct Contact
In order to evaluate of the ursolic acid and its derivatives as modulators of antibiotic resistance, the
MICs values of aminoglycosides antibiotics neomycin, kanamycin, amikacin, and gentamicin against
the analyzed strains were determined in the presence or absence of the extracts using the microdutiltion
test. Subinhibitory concentrations (MIC 1/8) in 10% BHI were used. The antibiotic solutions
(
1024 μg/mL) were prepared in distillated water for use on the same day. A total of 100 μL of the
antibiotic solution, using serial dilutions (1:2), was added to the wells containing 10% BHI and the
diluted bacterial suspension (1:10). Microplates were incubated at 35 ± 2 °C for 24 h and the
antibacterial activity was determined as described before [25].
3
.8. Antioxidant Activity
The antioxidant activities of ursolic acid (1), and derivatives 1a and 1b were evaluated by
measuring the reduction of the free radical 1,1-diphenyl-1-picrylhydrazyl (DPPH). The samples (1.0 to
000.0 μg/mL) were dissolved in methanol and then added to a methanol solution of DPPH (60 mM) [26].
1
After 30 min, the UV absorbance of the resulting solutions was recorded at λ 517 nm. The experiment
was performed in triplicate and the average absorption was noted for each concentration. Trolox and
Vitamin C were used as the positive control. The free radical scavenging activity was calculated as a

Molecules 2014, 19
1325
percentage inhibition of the DPPH radical by the sample or positive control. The IC value is the
5
0
concentration required to scavenge 50% DPPH.
. Conclusions
This study showed that ursolic acid (1) and some of its derivatives have significant antibacterial
4
activity against several bacterial species, and that these compounds show synergistic activities with the
aminoglycoside antibiotics neomycin, amikacin, kanamycin and gentamicin. These results suggest that
Sambucus australis could be a potential natural source of free radical scavengers.
Acknowledgments
The authors thank the Brazilian agencies CAPES and CNPq for fellowships and financial support.
Author Contributions
Patricia Nascimento, Ayla Bizerra: chromatography procedures and synthesis of derivatives;
Jose G. Costa: antimicrobial activity; Daniela Ferreira: antioxidant activity; R. Braz-Filho: analysis of
spectroscopic data; Telma Lemos, Gilvandete Santiago, advisors and mentors; Angela Arriaga: criticism.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples 1, 1a and 1b are available from the authors.
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