Phytochemistry Letters 11 (2015) 1823

Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

A new benzoic acid derivative from Piper diospyrifolium and its

anti-Mycobacterium tuberculosis activity
Regiane B. de L. Scodro a,f, Suelem C. Espelho a, Claudia T. Agostinho Pires b,
Vitor A. dos S. Garcia c, Lucio Cardozo-Filho d, Lucia E.R. Cortez e, Eduardo J. Pilau f,
Katiany Rizzieri Calef Ferracioli g, Vera Lucia D. Siqueira a,g, Rosilene F. Cardoso b,g,
Diogenes A.G. Cortez a,h,*
a

Postgraduate Program in Pharmaceutical Sciences, State University of Maringa (UEM), Av. Colombo, 5790, CEP 87020-900 Maringa, Parana, Brazil
Postgraduate Program in Biosciences Applied to Pharmacy, UEM, 87020-900 Maringa, Parana, Brazil
c
Department of Agronomy, UEM, 87020-900 Maringa, Parana, Brazil
d
Department of Chemical Engineering, UEM, 87020-900 Maringa, PR, Brazil
e
Postgraduate Program in Health Promotion, Cesumar, 87050-390 Maringa, Parana, Brazil
f
Department of Chemistry, UEM, 87020-900 Maringa, Parana, Brazil
g
Department of Clinical Analysis and Biomedicine, UEM, 87020-900 Maringa, Parana, Brazil
h
Department of Pharmacy, UEM, 87020-900 Maringa, Parana, Brazil
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 7 May 2013
Received in revised form 8 October 2014
Accepted 14 October 2014
Available online 25 October 2014

The extraction by supercritical uid (SFE-CO2) from leaves of Piper diospyrifolium and chromatographic
column purication afforded the isolation of a new benzoic acid derivative 4-methoxy-3-[(E)-3-methyl1,3-butadien-1-yl]-5-(3-methyl-2-buten-1-yl)-benzoic acid (1). The chemical structure was elucidated
on the basis of spectroscopic methods and comparison with literature data. SFE-CO2 extracts and (1)
were tested for their anti-Mycobacterium tuberculosis activities and cytotoxicities in J774G.8
macrophages. The compound (1) and SFE-CO2 extracts exhibited moderate activities against M.
tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 125 mg/mL. The MIC values of
M. tuberculosis clinical isolates ranged from 125 mg/mL to >250 mg/mL. The cytotoxicities results
showed a selectivity index range from 0.6 to 1.0. Additional studies in structure activity-relationship as
well as synergistic activity with antituberculous drugs should be conducted for a better evaluation of
anti-mycobacterial activity of this compound.
2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords:
SFE
Piper diospyrifolium
Benzoic acid derivative
Mycobacterium tuberculosis
Cytotoxicity

1. Introduction
Tuberculosis (TB) is a public-health issue of major signicance.
A total of 8.6 million cases and 1.3 million deaths from TB were
estimated for 2012. TB is the second leading cause of death from
infectious disease worldwide (WHO, 2013). Brazil is among the
22 countries that account for 80% of world TB cases, with 82,755
total cases notied in 2012 (WHO, 2013). This disease was
exacerbated by the AIDS pandemic and also by the emergence of
multidrug-resistant strains (Cantrell et al., 1998). These problems
have led to an intensied search for new classes of antimyco-

* Corresponding author at: Universidade Estadual de Maringa, Departamento de

Farmacia, Bloco B-49, Avenida Colombo, 5790, CEP 87020-900 Maringa, PR, Brazil.
Tel.: +55 44 3011 5248; fax: +55 44 3011 5248.
E-mail address: dagcortez@uem.br (Diogenes A.G. Cortez).

bacterial agents. In spite of the great advances in modern medicine

in recent decades, plants still make an important contribution to
health care (Luize et al., 2006).
Members of the family Piperaceae, which are widely distributed in
the tropics and subtropics, consist of about 10002000 species
(Monzote et al., 2010), and are used medicinally for several purposes
(Parmar et al., 1997). In Brazil, there are ve genera and
approximately 500 species of Piperaceae (Souza and Lorenzi,
2008). Piper diospyrifolium is a shrub, often forming small thickets
in the forest. Although P. diospyrifolium is common in forest remnants
in the northwestern of the State of Parana, Brazil, it has been relatively
little studied (Souza et al., 2004). The search for active constituents
from different Piper species has been intensied in recent years
(Pessini et al., 2005). Chemical studies carried out on Piperaceae
species have revealed the presence of several compounds including
alkaloids/amides, propenylphenols, lignans, neolignanas, terpenes,
steroids, kawapyrones, piperolides, chalcones, dihydrochalcones,

http://dx.doi.org/10.1016/j.phytol.2014.10.015
1874-3900/ 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

R.B.L. Scodro et al. / Phytochemistry Letters 11 (2015) 1823

2. Results and discussion

2.1. Supercritical uid extraction
To date, the extraction of bioactive compounds from P.
diospyrifolium leaves has not been carried out using extraction
of supercritical uid. Table 1 presents the experimental conditions
and extraction yields obtained from the P. diospyrifolium leaves
using SFE-CO2. The results indicate that yields were highest for the
25 MPa and 40 8C experimental conditions (2.88%). The pressure
was the factor that most inuenced the process, as shown by runs
3 and 4, which were carried out at 25 MPa. Pressure and
temperature are the two main factors affecting SFE. Lemos et al.
(2012) extracted neolignans from P. regnellii using SFE-CO2, and
observed that for a constant temperature, an increase in pressure
enhances the yield, because increasing pressure at a constant
temperature will increase the density and dissolving capacity of
supercritical CO2.
On the other hand, in extracting alkaloids from P. amalago using
SFE, Carrara et al. (2012) observed that a higher yield occurred at

Table 1
Conditions of supercritical uid extraction with carbon dioxide as solvent, used to
produce extracts from Piper diospyrifolium leaves.
Run
1
2
3
4
a

Pressure
(MPa)

Temperature
(8C)

Solvent
density (g/mL)

Yield (%)a

14
14
25
25

20
40
20
40

0.89
0.78
0.96
0.88

1.12
1.27
1.51
2.88

Yield (%) = (extracted mass/mass of dry sample)  100.

3.0
Run 1
Run 2
Run 3
Run 4

2.5

2.0
Yield [%]

avones, avanones, and miscellaneous compounds including

benzoic acid derivatives (Parmar et al., 1997). Although there are
many reports on the phytochemistry of the genus Piper, we know
of no previous study of the chemical constituents and antimycobacterial activity of P. diospyrifolium. Benzoic acid derivatives are
frequently isolated from Piper species, and many of them exhibit
diverse biological activities, such as antimicrobial, molluscicidal
(Orjala et al., 1993), antifungal (Terreaux et al., 1998) and
leishmanicidal (Flores et al., 2009).
The conventional process for extraction of natural compounds
used in medicine and industries is laborious, involving the use of
large volumes of organic reagents that may cause deleterious
environmental effects. Additional problems may occur from the
presence of traces of toxic solvents in the extract and degradation
of thermo-labile compounds (Doker et al., 2010; Almeida et al.,
2012). Since the end of the 1970s, supercritical uid extraction
(SFE) has been used to isolate natural products. SFE is a versatile
technology, based mainly on varying the pressure, temperature,
density and solvent used during the procedure, to obtain good
yields of clean extracts with short extraction times (Meireles,
2009). Carbon dioxide is widely used as a solvent in SFE, mainly
due to its physical and chemical properties, such as low critical
pressure (7.382 MPa) and temperature (31 8C). The use of SFE with
carbon dioxide (SFE-CO2) as the organic solvent to obtain bioactive
compounds has many advantages compared to the conventional
process, especially in the food, pharmaceutical and cosmetic
industries. Carbon dioxide is the standard solvent of choice for
SFE of natural products, because it is inert and behaves as a
lipophilic solvent (McHugh and Krukonis, 1986). The present study
determined the anti-Mycobacterium tuberculosis activities of
extracts obtained by SFE-CO2 and a benzoic acid derivative
obtained from P. diospyrifolium leaves, as well as the cytotoxicities
of these substances.

19

1.5

1.0

0.5

0.0
0

20

40

60

80

100

120

140

Time [min]

Fig. 1. Kinetic curves of supercritical uid extractions from Piper diospyrifolium

leaves using SFE-CO2. Conditions of extraction: [*] 14 MPa, 20 8C; [&] 14 MPa,
40 8C; [^] 25 MPa, 20 8C; [D] 25 MPa, 40 8C.

lower pressure and that SFE-CO2 was more effective than

compressed propane. Fig. 1 shows the kinetic curves of the
extraction process of P. diospyrifolium leaves using SFE-CO2;
clearly, an increase in pressure improved the extraction rates.
These results are in agreement with other studies (Moura et al.,
2009; Prado et al., 2011; Lemos et al., 2012).
2.2. Purication and identication of SFE-CO2 extracts
The purication of SFE-CO2 extracts from P. diospyrifolium
leaves yielded a new benzoic acid derivative named as 4-methoxy3-[(E)-3-methyl-1,3-butadien-1-yl]-5-(3-methyl-2-buten-1-yl)benzoic acid (1) (Fig. 1). SFE-CO2 was effective for extracting this
compound. The molecular formula of (1) was determined to be
C18H22O3 by analysis of its EI-MS [m/z: 286 (100)] and NMR data
(Table 2). The splitting pattern in the 1H NMR spectrum (300 MHz)
of the two aromatic protons [dH 8.15 (d; J = 1.8 Hz)] and [dH 7.81
Table 2
1
H13C NMR data of the studied compound (1) 4-methoxy-3-[(E)-3-methyl-1,3butadien-1-yl]-5-(3-methyl-2-buten-1-yl)-benzoic acid obtained from Piper diospyrifolium (CDCl3, 300 MHz) compared to 13C NMR of compound (2) 3-(2-hydroxy3-methyl-3-buten-1-yl)-4-methoxy-5-(3-methyl-2-buten-1-yl)-benzoic acid obtained
(CDCl3, 400 MHz) by Flores et al. (2008).
Position

Compounds
(2)

(1)
( d C)
1
2
3
4
5
6
10
20
30
40

125.1
126.8
135.7
160.4
134.2
130.9
122.5
133.9
142.1
118.3

50
100
200
300
400
500
OMe
COOH

18.6
28.4
122.1
131.2
17.9
25.8
61.4
171.2

13

H C HSQC
(dH) (J in Hz)

8.15 (d; 1.8)

H-6
H-10
OMe; H-2; H-6; H-10 ; H-100
H-100 ; H-200
H-2
H-20 ; H-2
HA-40 ; HB-40 ; H-50
H-10 ; H-20 ; H-50
H-20
H-20 ; H-50

7.81 (d; 1.8)

6.79 (d; 16.5)
6.98 (d; 16.5)
A: 5.18; sl
B: 5.13; sl
2.00; s
3.39 (d; 7.2)
5.29 (t; 7.2)
1.75; s
1.75; s
3.78; s

13

H C HMBC

00

H-1 ; H-4
H-200
H-400
H-6; H-2

00

( d C)
124.8
130.9
131.9
161.1
135.2
130.9
36.5
75.8
146.8
110.9
17.8
28.0
121.8
130.4
17.7
25.5
60.8
170.1

R.B.L. Scodro et al. / Phytochemistry Letters 11 (2015) 1823

20

Table 3
Anti-Mycobacterium tuberculosis (H37Rv) activity and cytotoxicities of extracts and
benzoic acid derivative from Piper diospyrifolium leaves.

OH

SFE-CO2 extracts and compound

Pressure (MPa)

1
5

4''
3''

2'

4'

1''

3'

OC H3

5''

Temperature (8C)

20
14
14
40
20
25
25
40
Benzoic acid derivative

MIC (mg/mL)

IC50

SI

125
125
125
125
125

101.0
116.0
77.5
82.5
126

0.8
0.9
0.6
0.7
1.0

IC50, 50% cytotoxic concentration for the macrophages J774.G8; MIC, minimum
inhibitory concentration; SI, selectivity index.

5'

Fig. 2. Correlation of HMBC of compound 1.

(d; J = 1.8 Hz)] established that the aromatic ring was a 1,3,4,5tetrasubstituted benzene ring. In the HMBC spectrum of (1)
(Table 2 and Fig. 2), the proton signal at dH 8.15 (H-2) and 7.81 (H6) showed cross-peaks to a carbonyl carbon at dC 171.2 and a
quaternary carbon at dC 160.4 (C-4). The correlation for methyl
proton [dH 3.78 (s)] with C-4 indicated the location of the
methoxyl group shown by the HSQC (heteronuclear single
quantum correlation) and HMBC (heteronuclear multiple-bond
correlation) experiments. Additional signals included two vinyl
methyl groups as singlets at d 1.75 and 2.00 with one to nine
protons, one aliphatic methylene at d 3.39 (d; J = 7.2 Hz), one
methine at d 5.29 (t; J = 7.2 Hz), two olen at d 6.79 and 6.98 (1H
each, d) having a large coupling constant (J = 16.5 Hz) were
attributable to an (E)-disubstituted double bond (Ito and
Furukawa, 1987; Ito et al., 2000) and two of them as large
singlets at d 5.13 and 5.18, showing the presence of three double
bonds in the side chain. The 13C NMR (Table 2) spectrum exhibited
signals of 18 carbons. These data indicated that (1) was identied
as 4-methoxy-3-[(E)-3-methyl-1,3-butadien-1-yl]-5-(3-methyl2-buten-1-yl)-benzoic acid, similar to those of compound (2)
(Fig. 3) (Flores et al., 2008). The difference was conrmed by the
presence of the signals at dC 36.5 and 75.8, corresponding to a
methylene and one oxymethine carbon in the side chain.
2.3. Anti-M. tuberculosis activity assay
The in vitro anti-M. tuberculosis activity of SFE-CO2 extracts and
the benzoic acid derivative (1) from P. diospyrifolium leaves was
evaluated using REMA. All tested SFE-CO2 extracts showed
moderate activity against M. tuberculosis H37Rv ATCC 27294 with
MIC values of 125 mg/mL (Table 3). The operating conditions used
to obtain the SFE-CO2 extracts did not affect the anti-M.
tuberculosis activities, since their MICs remained the same for all

conditions tested, probably because these extracts contained the

same compounds with activity against the bacillus.
Tosun et al. (2004) working with M. tuberculosis H37Ra,
considered an extract or compounds that prevent bacillus growth
up to a concentration of 200 mg/mL as a good anti-M. tuberculosis
drug candidate. Other authors have considered values such as
MIC  128 and 64 mg/mL to indicate activity against M. tuberculosis H37Rv for extracts and pure compounds, respectively (Cantrell
et al., 2001; Gu et al., 2004).
Because the mycobacteria cell wall contains a large amount of
mycolic acid (lipophilic) (Palomino et al., 2002), the benzoic acid
derivative was tested. Despite its non-polar structure, did not
change in the MIC value against M. tuberculosis (Table 3). The
lipophilicity of compounds is an important feature to consider for
antimycobacterial activity of compounds, as reported by Castellar
et al. (2011) since this improves the penetration of substances into
the cell.
The benzoic acid derivative was also tested against clinical
isolates (susceptible and resistant) (Table 4). The MICs for the
clinical isolates ranged from 125 mg/mL to >250 mg/mL. The
biological effect of the benzoic acid derivative has been previously
reported, but not against M. tuberculosis. Terreaux et al. (1998)
isolated four benzoic acid derivatives from leaves of P. dilatatum,
which displayed antifungal activity against Cladosporium cucumerinum by a bioautographic test, with MICs from 1 to 5 mg/mL.
The activity of other species of Piper against M. tuberculosis has
been studied. Rukachaisirikul et al. (2002) isolated a novel piperine
dimer (chabamide) from stems of P. chaba, which showed an MIC of
12.5 mg/mL against M. tuberculosis H37Ra strain. In a review of
antitubercular natural products (Garca et al., 2012), during the last
ve years, only one species of Piper (P. sarmentosum) was found to
exhibit moderate inhibitory activity against M. tuberculosis H37Ra,
with MICs from 25 to 50 mg/mL.
The selectivity index (SI) of each compound was determined as
the IC50 to MIC ratio in J774G.8 cell line macrophage (Table 3). The

OH

3''

2'
OCH3

3''

1''
5''

OH

1
4''

OH

3'

4'

3
2'

1''
OCH3

3'
5'

Fig. 3. Structures of 4-methoxy-3-[(E)-3-methyl-1,3-butadien-1-yl]-5-(3-methyl-2-buten-1-yl)-benzoic acid (1), a new benzoic acid derivative from Piper diospyrifolium
leaves and 3-(2-hydroxy-3-methyl-3-buten-1-yl)-4-methoxy-5-(3-methyl-2-butenyl)-benzoic acid (2) (Flores et al., 2008).

R.B.L. Scodro et al. / Phytochemistry Letters 11 (2015) 1823

Table 4
Minimum inhibitory concentration (MIC) of the benzoic acid derivative (1) from
Piper diospyrifolium leaves against Mycobacterium tuberculosis strains.
Clinical isolates

Resistance

MIC (mg/mL)

49
13638
4851
57
17
91
73-A
3614

Susceptible
Susceptible
Susceptible
Susceptible
H
H, S
H, R, Z
H, R, E, S, Et

250
250
>250
250
250
>250
>250
>250

H, isoniazid; R, rifampicin; Z, pyrazinamide; E, ethambutol; Et, etionamide; S,

streptomycin.

SFE-CO2 extracts and pure compounds showed an SI range from

0.6 to 1.0. SI is used to estimate the therapeutic window of a drug
and to identify drug candidates for further studies (Pavan et al.,
2011) and candidates for new drugs must have an SI equal to or
higher than 10 (Garca et al., 2012; Orme, 2001). Because all the
SFE-CO2 extracts and pure compounds studied here exhibited no
therapeutic selectivity, a future line of research is to study their
action against cancerous tumors and the structureactivity by
changing radicals in the benzoic acid derivative. This is the rst
report that shows a benzoic acid derivative (1) from P.
diopyrifolium leaves, as well as its anti-M. tuberculosis activity.
SFE-CO2 has proven to be an excellent tool for extraction of natural
products, with good yields and with fewer undesirable components. Additional studies in mycobacteria other than tuberculosis
(MOTT) using extracts and the benzoic acid derivative (1), as well
as studies of structureactivity and the synergistic activity with
other antituberculous drugs, should be conducted for a better
evaluation of the anti-mycobacterial activity.
3. Experimental
3.1. General experimental procedures
The spectra were obtained in a Varian Gemini 300 (7.05 T),
using TMS as the internal standard. EI-MS on a GC/MS-Thermo QP
2000 A. CC: silica gel 60 (70230 and 230400 mesh); TLC: silica

21

gel plates F254 (0.25 mm thick). A Waters Synapt HDMS QTOF Mass
Spectrometer (Waters, Milford, MA, USA), with an electrospray
ionization source (ESI) was used to perform ESI-MS and ESI-MS/
MS, in negative mode, experiments. The mass spectrometer
operating conditions were: capillary voltage at 3.0 kV; cone
voltage was 30 V. The source temperature and desolvation gas
temperature were set at 100 and 200 8C, respectively. The cone gas
ow (L/h) and desolvation gas ow (L/h) were 10 and 900,
respectively. For ESI-MS/MS experiments the Trap Collision Energy
was maintained in 10. The TOF was operated in W mode.
3.2. Plant material
Leaves of P. diospyrifolium were collected in October 2010 in the
Maringa Forest Park, State of Parana, Brazil. A voucher specimen is
deposited in the herbarium of the Department of Botany, State
University of Maringa (HUM 9392).
3.3. Raw material preparation
P. diospyrifolium leaves were dried and shredded in a knife mill
(Tecnal TE-631).
3.4. Supercritical uid extraction procedures
The experiments were performed in a bench-scale unit (Fig. 4),
in the Department of Chemical Engineering, State University of
Maringa. This unit consists basically of a compressed-gas reservoir
(carbon dioxide, technical grade, obtained from White Martins,
Brazil), two thermostatic baths, two syringe pumps (Teledyne ISCO
500D, Lincoln, NE, USA), and a 166.5 cm3 jacketed extraction
vessel. In each experiment, the extractor was loaded with
approximately 25 g of powdered sample. Compressed CO2 was
pumped into the bed of Piper powder at a constant ow rate of
3 mL/min for CO2. After 10 min, the extraction was interrupted for
measurement of the extracted mass; and this procedure was
repeated for 120 min. The CO2 extraction experiments were
conducted at temperatures from 20 to 40 8C and pressures from
14 to 25 MPa. The solvent density was calculated according to
Angus et al. (1976). Runs were performed in duplicate for all
conditions.

Fig. 4. Experimental apparatus: C1, jacketed extraction vessel; C2, solvent reservoir; V1, metering valve; V2, needle valve; A and B, syringe pump; PC-1 and PC-2, pump
control; PT, pressure transmitter; TT, temperature transmitter; TB-1 and TB-2, thermostatic baths.

22

R.B.L. Scodro et al. / Phytochemistry Letters 11 (2015) 1823

3.5. Purication of SFE-CO2 extracts

3.7. Cytotoxicity assay

All the SFE-CO2 extracts (387.2 g) were chromatographed on a

silica gel 60 (70230 mesh) column, eluted with hexane (100 mL),
hexane/EtOAc (7:3, v/v, 30 mL) hexane/EtOAc (1:1, v/v, 30 mL),
EtOAc (15 mL) and methanol (15 mL), affording 17 fractions.
Fraction F9 (69 mg) was identied as a benzoic acid derivative
named 4-methoxy-3-[(E)-3-methyl-1,3-butadien-1-yl]-5-(3-methyl-2-buten-1-yl)-benzoic acid (1), by analyses of spectral data of 1H
and 13C and by comparison with literature data (Flores et al., 2008)
(Table 2).

In vitro cytotoxicity assays were performed on the J774G.8

macrophage (Skehan et al., 1990; Papazisis et al., 1997). The
macrophage was maintained in tissue asks containing RPMI
(Gibco, Invitrogen Co., Grand Island, NY, USA) with L-glutamine and
supplemented with 10% inactivated fetal bovine serum (FBS)
(Gibco BRL, Life Technologies) at 37 8C in 5% CO2 air atmosphere.
Macrophages were plated at 5.0  104 cells per well in a 96-well
plate with RPMI 1640 medium supplemented with 10% inactivated
FBS, and incubated for 24 h at 37 8C in 5% CO2. Then, the medium
was removed and the cell monolayers were treated with different
concentrations of the extracts (12.5250 mg/mL). Positive (with
Isoniazid) and negative controls (without drugs) were included.
The plate was incubated in the same conditions for 48 h.
Subsequently, the sulforhodamine B (SRB) (Sigma Chemical Co.)
colorimetric assay was carried out. The cells were xed by adding
10% trichloroacetic acid (TCA) and incubating for 1 h at 4 8C. The
plates were washed with distilled water, air-dried, and stained
with SRB solution (0.4%, w/v in 1% acetic acid) at 4 8C for 30 min.
Cells were washed four times with 1% acetic acid. The bound SRB
stain was solubilized with 150 mL of 10 mM Tris buffer (Sigma).
The optical density was determined using an automated
spectrophotometer (Asys Expert Plus) at 530 nm. The 50%
cytotoxicity concentration (IC50) was determined by logarithm
regression analysis. The selectivity index (SI) was determined by
the IC50/MIC ratio for each compound tested.

3.5.1. Compound (1)

4-Methoxy-3-[(1E)-3-methyl-1,3-butadien-1-yl]-5-(3-methyl2-buten-1-yl)-benzoic acid: semi-solid. 1H NMR 1 and 13C NMR:
see Table 2. EI-MS, m/z (rel. int.): 286(100), 271(32), 240(30),
209(32), 198(81), 172(30). HR-MS (ESI) [MH] found m/z:
285.1489 (calc. for C18H21O3 [MH] 285.1491).
3.6. Anti-M. tuberculosis activity assay
3.6.1. Reference drug
Isoniazid (Difco laboratories, Detroit, MI, USA) solution was
prepared at a concentration of 4 mg/mL in distilled water, ltersterilized, and frozen until used. Just before use, an initial working
concentration of 4 mg/mL was prepared in Middlebrook 7H9
(Difco) with added oleic acid-albumin-dextrose-catalase (OADC)
enrichment (BBL/Becton-Dickinson, Sparks, MD, USA).

Acknowledgments
3.6.2. Mycobacterial reference strain and clinical isolates
M. tuberculosis H37Rv (ATCC 27294) and eight M. tuberculosis
clinical isolates (four susceptible and four resistant) from the
mycobacteria collection of the Laboratory of Medical Bacteriology
of the State University of Maringa, Maringa, Parana, Brazil, were
employed for the antimycobacterial assay.
3.6.3. Minimal inhibitory concentration
The anti-M. tuberculosis activities of the SFE-CO2 extracts and
benzoic acid derivative from leaves of P. diospyrifolium were
evaluated by colorimetric resazurin microtiter assay plate
(REMA) as described by Palomino et al. (2002) and Martin et al.
(2003), with modications. Briey, 200 mL of sterile distilled
water was distributed in the outer wells of the microplate
(Falcon 3072, Becton Dickinson, Lincoln Park, NJ, USA); extracts
and pure compound were diluted in dimethyl sulfoxide (DMSO)
(Amresco), and serial twofold dilutions from 250 to 1.9 mg/mL
were made in Middlebrook 7H9 medium supplemented with
OADC. Isoniazid was used as the reference drug at concentrations
from 1.0 to 0.007 mg/mL. One hundred microliters of each
bacterial inoculum, standardized according to 1 McFarland
turbidity and diluted 1:20 in Middlebrook 7H9 medium supplemented with OADC, was inoculated into the wells containing
media. A growth control containing no drug and a sterile
control without bacteria were also prepared for each assay.
The plates were covered with their lids and their edges
sealed with polyethylene tape. The plates were placed in a
plastic box and incubated at 35 8C at normal atmosphere
for 7 days. Then, 30 mL of freshly prepared 0.01% resazurin
solution (Acros, NJ, USA) was added to each well and the
plates were reincubated at 35 8C for 24 h for visual readings.
A color change from blue to pink indicated mycobacterial
growth, and the MIC was interpreted as the lowest extract
concentration that prevented the color change. A control containing 2.5% (v/v) DMSO was included to detect inhibitory effects
on M. tuberculosis growth. All experiments were performed in
triplicate.

The authors are grateful to CNPq, CAPES, and Fundacao

Araucaria for the nancial support. We are thankful to the
Laboratory of Clinical Immunology of the State University of
Maringa, Brazil where the cytotoxicities assays were performed.
We thank Janet W. Reid for revising the English text.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2014.10.015.
References
Almeida, P.P., Mezzomo, N., Ferreira, S.R.S., 2012. Extraction of Mentha spicata L.
volatile compounds: evaluation of process parameters and extract composition.
Food Bioprocess. Technol. 5, 548559.
Angus, S., Armstrong, B., Reuck, K.M., 1976. International Thermodynamic Tables of
the Fluid State. Carbon Dioxide. Pergamon Press, New York.
Cantrell, C.L., Fischer, N.H., Urbatsch, L., McGuire, M.S., Franzblau, S.G., 1998.
Antimycobacterial crude plant extracts from South, Central, and North America.
Phytomedicine 5, 137145.
Cantrell, C.L., Franzblau, S.G., Fischer, N.H., 2001. Antimycobacterial plant terpenoids. Planta Med. 67, 110.
Carrara, V.S., Serra, L.Z., Cardozo-Filho, L., Cunha-Junior, E.F., Torres-Santos, E.C.,
Cortez, D.A.G., 2012. HPLC analysis of supercritical carbon dioxide and compressed propane extracts from Piper amalago L. with antileishmanial activity.
Molecules 17, 1533.
Castellar, A., Coelho, T.S., Silva, P.E.A., 2011. The activity of avones and oleanolic
acid from Lippia lacunosa against susceptible and resistant Mycobacterium
tuberculosis strains. Rev. Bras. Farmacogn. 21, 835840.
Doker, O., Salgin, U., Yildiz, N., Aydogmus, M., Calimli, A., 2010. Extraction of sesame
seed oil using supercritical CO2 and mathematical modeling. J. Food Eng. 97,
360366.
Flores, N., Jimenez, I.A., Gimenez, A., Ruiz, G., Gutierrez, D., Bourdy, G., Bazzocchi, I.,
2008. Benzoic acid derivatives from Piper species and their antiparasitic activity. J. Nat. Prod. 71, 15381543.
Flores, N., Jimenez, I.A., Gimenez, A., Ruiz, G., Gutierrez, D., Bourdy, G., Bazzocchi, I.,
2009. Antiparasitic activity of prenylated benzoic acid derivatives from Piper
species. Phytochemistry 70, 621627.
Garca, A., Bocanegra-Garca, V., Palma-Nicolas, J.P., Rivera, G., 2012. Recent
advances in antitubercular natural products. Eur. J. Med. Chem. 49, 123.

R.B.L. Scodro et al. / Phytochemistry Letters 11 (2015) 1823

Gu, J.Q., Wang, Y., Franzblau, S.G., 2004. Antitubercular constituents of Valeriana
laxiora. Planta Med. 70, 509514.
Ito, C., Furukawa, H., 1987. Constituents of Murraya exotica L. structure elucidation
of new coumarins. Chem. Pharm. Bull. 35, 42774285.
Ito, C., Otsuka, T., Ruangrungsi, N., Furukawa, H., 2000. Chemical constituents of
Micromelum minutum. Isolation and structural elucidation of new coumarins.
Chem. Pharm. Bull. 48, 334338.
Lemos, C.O.T., Garcia, V.A.S., Goncalves, R.M., Leal, I.C.R., Siqueira, V.L.D., CardozoFilho, L., Cabral, V.F., 2012. Supercritical extraction of neolignans from Piper
regnellii var. pallescens. J. Supercrit. Fluids 71, 6470.
Luize, P.S., Ueda-Nakamura, T., Dias Filho, B.P., Cortez, D.A.G., Morgado-Daz, J.A.,
Souza, W., Nakamura, C.V., 2006. Ultrastructural alterations induced by the
neolignan dihydrobenzofuranic eupomatenoid-5 on epimastigote and amastigote forms of Trypanosoma cruzi. Parasitol. Res. 100, 3137.
Martin, A., Camacho, M., Portaels, F., Palomino, J.C., 2003. Resazurin microtiter assay
plate testing of Mycobacterium tuberculosis susceptibilities to second-line
drugs: rapid, simple, and inexpensive method. Antimicrob. Agents Chemother.
47, 36163619.
McHugh, M., Krukonis, V., 1986. Supercritical Fluid Extraction Principles and
Practice. Butterworth Publishers, Stoneham.
Meireles, M.A.A., 2009. Extracting Bioactive Compounds for Food Products: Theory
and Application, rst ed. CRC Press Taylor and Francis Group, Boca Raton.
Monzote, L., Garca, M., Montalvo, A.M., Scull, R., Miranda, M., 2010. Chemistry,
cytotoxicity and antileishmanial activity of the essential oil from Piper auritum.
Mem. Inst. Oswaldo Cruz 105, 168173.
Moura, L.S., Favareto, R., Leal, P.F., Corazza, M.L., Cardozo-Filho, L., Meireles, M.A.A.,
2009. Phase-equilibrium measurements for CO2 + priprioca extract at high
pressures. J. Supercrit. Fluids 48, 126130.
Orjala, J., Erdelmeyer, C.A.J., Wright, A.D., Rali, T., Sticher, O., 1993. Two chromenes
and a prenylated benzoic acid derivative from Piper aduncum. Phytochemistry
34, 813818.
Orme, I., Tuberculosis Drug Screening Program, 2001. Search for new drugs for
treatment of tuberculosis. Antimicrob. Agents Chemother. 45, 19431946.
Palomino, J.C., Martin, A., Camacho, M., Guerra, H., Swings, J., Portaels, F., 2002.
Resazurin microtiter assay plate: simple and inexpensive method for detection

23

of drug resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 46, 27202722.

Papazisis, K.T., Geromichalos, G.D., Dimitriadis, K.A., Kortsaris, A.H., 1997. Optimization of the sulforhodamine B colorimetric assay. J. Immunol. Methods 208,
151158.
Parmar, V.S., Jain, S.C., Bisht, K.S., Jain, R., Taneja, P., Jha, A., Tyagi, O.D., Prasad, A.K.,
Wengel, J., Olsen, C.E., Boll, P.M., 1997. Phytochemistry of the genus Piper.
Phytochemistry 46, 597673.
Pavan, F.R., Poelhsitz, G.V., Barbosa, M.I.F., Leite, S.R.A., Batista, A.A., Ellena, J., Sato,
L.S., Franzblau, S.G., Moreno, V., Gambino, D., Leite, C.Q.F., 2011. Ruthenium (II)
phosphine/diimine/picolinate complexes: inorganic compounds as agents
against tuberculosis. Eur. J. Med. Chem. 46, 50995107.
Pessini, G.L., Dias Filho, B.P., Nakamura, C.V., Cortez, D.A.G., 2005. Antifungal activity
of the extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens
(C. DC.) Yunck. J. Braz. Chem. Soc. 16, 11301133.
a,
Prado, I.M., Giufrida, W.M., Alvarez, V.H., Cabral, V.F., Quispe-Condori, S., Saldan
M.D.A., Cardozo-Filho, L., 2011. Phase equilibrium measurements of sacha inchi
oil (Plukenetia volubilis) and CO2 at high pressures. J. Am. Oil Chem. Soc. 88,
12631269.
Rukachaisirikul, T., Prabpai, S., Champung, P., Suksamrarn, A., 2002. Chabamide, a
novel piperine dimer from stems of Piper chaba. Planta Med. 68, 853855.
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahom, J., Vistica, D., 1990. New
colorimetric cytotoxicity assays for anticancer-drug screening. J. Natl. Cancer
Inst. 82, 11071112.
Souza, L.A., Moscheta, I.S., Oliveira, J.H.G., 2004. Comparative morphology and
anatomy of the leaf and stem of Peperomia dahlstedtii C.DC., Ottonia martiana
Miq. and Piper diospyrifolium Kunth (Piperaceae). Gayana Bot. 61, 617.
Souza, V.C., Lorenzi, H., 2008. Botanica sistematica: guia ilustrado para identicacao
das famlias de fanerogamas nativas e exoticas no Brasil, baseado na APG II,
second ed. Instituto Plantarum, Sao Paulo.
Terreaux, C., Gupta, M.P., Hostettmann, K., 1998. Antifungal benzoic acid derivatives
from Piper dilatatum. Phytochemistry 49, 461464.
Tosun, F., Kizilay, C.A., Sener, B., Vural, M., Palittapongarnpim, P., 2004. Antimycobacterial screening of some Turkish plants. J. Ethnopharmacol. 95, 273275.
WHO, 2013. Global Tuberculosis Report 2013. WHO Press, Geneva.