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Journal of Ethnopharmacology 150 (2013) 655–664

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Inhibition of the human neutrophil oxidative metabolism by Baccharis


dracunculifolia DC (Asteraceae) is influenced by seasonality and the
ratio of caffeic acid to other phenolic compounds
Andréa S.G. Figueiredo-Rinhel a, Luciana M. Kabeya a, Paula C.P. Bueno b,1,
Renata F. Jorge-Tiossi c, Ana Elisa C.S. Azzolini a, Jairo K. Bastos c,
Yara Maria Lucisano-Valim a,n
a
Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café s/n, Monte Alegre,
14040-903 Ribeirão Preto, SP, Brazil
b
Laboratório de Pesquisa, Desenvolvimento & Inovação, Apis Flora Indl. Coml. Ltda., Rua Triunfo 945, 14020-670 Ribeirão Preto, SP, Brazil
c
Laboratório de Farmacognosia, Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São
Paulo, Avenida do Café s/n, Monte Alegre, 14040-903 Ribeirão Preto, SP, Brazil

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: The great potential of phytotherapic drugs for treating and preventing
Received 18 June 2013 inflammatory diseases mediated by increased neutrophil reactive oxygen species (ROS) generation has
Received in revised form guided the search for new natural products with antioxidant and immunomodulatory properties.
12 September 2013
Baccharis dracunculifolia D.C. (Asteraceae), the main botanical source of Brazilian green propolis, is a
Accepted 17 September 2013
Available online 25 September 2013
native plant from Brazil widely used in folk medicine as anti-inflammatory. This study aims: (a) to
determine the influence of seasonality on the chemical profile and biological activity of Baccharis
Keywords: dracunculifolia (Asteraceae) leaf extracts (BdE); (b) to analyze the correlation between the major
Baccharis dracunculifolia compounds and the ability of BdE to modulate the superoxide anion and total ROS generation by
Antioxidant activity
human neutrophils.
Neutrophils
Materials and methods: The extracts were obtained from leaf samples collected monthly during one year.
Flavonoids
Phenolic compounds The superoxide anion and total ROS generation were assessed by the lucigenin (CL-luc)- and luminol
Seasonality (CL-lum)-enhanced chemiluminescence assays.
Results: Seasonality influenced more the quantitative than the qualitative chemical profile of B.
dracunculifolia, and affected its biological activity. The major compounds identified were caffeic acid,
p-coumaric acid, aromadendrin-4′-methyl ether (AME), isosakuranetin and artepillin C. The IC50 values
obtained for CL-lum and CL-luc inhibition by BdE ranged from 8.1–15.8 and 5.8–13.3 mg mL  1,
respectively, and correlated positively with caffeic acid concentration. CL-luc inhibition correlated
negatively with the concentration of artepillin C, AME, isosakuranetin and total flavonoids. The BdE
sample from May/07 inhibited CL-lum and CL-luc the most strongly (IC50 ¼8.1 7 1.6 and 5.87
1.0 mg mL  1, respectively), and contained the highest ratio of caffeic acid to the other isolated
compounds; so, this ratio could be employed as chemical marker for this biological activity of B.
dracunculifolia.
Conclusion: The ability of B. dracunculifolia to inhibit the neutrophil ROS generation depends more on the
type and ratio of phenolic compounds and flavonoids than on their high absolute concentrations.
Together, our results help select the most appropriate plant material for the production of phytotherapic
drugs to be used in the treatment of inflammatory diseases mediated by increased neutrophil activation.
& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Some chronic inflammatory diseases, in particular those mediated


n
by immune complexes, are associated with intense recruitment and
Corresponding author. Tel.: þ 55 16 3602 4212; fax: þ55 16 3602 4880.
activation of neutrophils in the tissues. The neutrophils play an impo-
E-mail address: yaluva@usp.br (Y.M. Lucisano-Valim).
1
Present Address: Instituto de Química, Universidade Estadual Paulista, Rua rtant role in host defense and in the regulation of innate and adaptive
Francisco Degni n. 55, 14800-900 Araraquara, SP, Brazil. immune responses. Once at the site of inflammation, activated

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.09.019
656 A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664

neutrophils secrete a variety of pro-inflammatory cytokines, of this variation in the biological effect of the extracts is not
eicosanoids and chemokines and release a large amount of reactive known. Thus, this study aims to determine the influence of
oxygen species (ROS) and proteolytic enzymes that can damage the seasonality on the ability of B. dracunculifolia to inhibit the
tissue and favor the development of pain and deformity of the target oxidative metabolism of human neutrophils as well as to analyze
organ. Rheumatoid arthritis, glomerulonephritis and vasculitis are the correlation between chemical composition and biological
some examples of immune complex-mediated inflammatory diseases activity of the extracts.
that affect a significant percentage of the human population and
diminish the quality of life and life expectancy (Kolaczkowska and
Kubes, 2013; Németh and Mócsai, 2012). 2. Material and methods
On the other hand, the production of adequate amounts of ROS by
neutrophils is essential to kill microbes and to keep the cellular and 2.1. Chemicals
tissue redox balance. This balance affects the physiological functions
of cells such as proliferation, chemotaxis and apoptosis, and is impo- Caffeic acid (3,4-dihydroxycinnamic acid), p-coumaric acid
rtant to regulate the course of acute and chronic inflammatory (4-hydroxycinnamic acid), lucigenin (N,N′-dimethyl-9,9′-biacridinium
responses, and autoimmune inflammation (Hultqvist et al., 2009). dinitrate), luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), quer-
Therefore, modulating this neutrophil function is an important cetin (3,3′,4′,5,7-pentahydroxyflavone dihydrate), Triton x-100, Trypan
therapeutic strategy to manage inflammation and reduce the dele- Blue, and Zymosan A were purchased from Sigma Chemical Co. (St.
terious effects of ROS. Louis, MO, USA). The other reagents used were: artepillin C (3,5-
Our research team has investigated the antioxidant and immu- diprenyl-4-hydroxycinnamic acid; Wako Pure Chemical Industries Co.,
nomodulatory properties of crude extracts such as green propolis, Osaka, Japan), dimethyl sulfoxide (DMSO; Merck, Darmstadt, Ger-
Tamarindus indica, Lychnophora sp, and Alternanthera maritima, as many), gallic acid (Labsynth, Diadema, SP, Brazil), gelatin of micro-
well as the properties of some phenolic compounds derived from biological grade (Difco Laboratories, Detroit, MN, USA), isosakuranetin
these natural products such as the flavonoids and coumarins (4′-methoxy-5,7-dihydroxyflavonone; ChromaDex, Irvine, Canada),
(Grael et al., 2010; Kanashiro et al., 2004; Paula et al., 2009; and LDH Liquiform Kit (Labtest Diagnostica, Lagoa Santa, MG, Brazil).
Simões et al., 2004; Souza et al., 2007). Among these products, we Aromadendrin-4′-methyl ether (AME) was isolated and identified by
found that Brazilian green propolis (BGP) is a promising modulator Sousa et al. (2007) and kindly donated by the authors. For quantitative
of the neutrophil ROS generation (Simões et al., 2004). However, analysis we used HPLC-grade methanol (Mallinckrodt Baker Inc, Paris,
the chemical composition of BGP varies a lot under the influence KY, USA) and deionized water (Milli-Q water purification system). All
of the season, botanical source, site of collection and bee species; the other chemicals were of reagent grade and were used without
this variability interferes in its biological properties and impairs further purification.
the standardization of BGP-derived products for medicinal pur-
poses (Sforcin and Bankova, 2011; Simões-Ambrósio et al., 2010). 2.2. Preparation of Baccharis dracunculifolia extracts (BdE)
Baccharis dracunculifolia D.C. (Asteraceae), commonly known as
“Alecrim do campo” and “Vassoura”, is the main botanical source of The B. dracunculifolia leaves were collected monthly in Cajuru
BGP. It is a native Brazilian plant widely used in folk medicine as anti- (São Paulo State, Brazil) from August 2006 to July 2007. Professor
inflammatory (Park et al., 2002; Simões et al., 2004). The traditional Nelson Ivo Matzenbacher authenticated the plant material, and a
use of B. dracunculifolia has been corroborated by its pharmacological voucher specimen (no. 1298) was deposited in the herbarium of
effects in vivo and in vitro experimental models. For instance, the Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e
B. dracunculifolia extracts show anti-inflammatory and analgesic Agronômicas (CPQBA) of Universidade Estadual de Campinas
effects in mice and rats (Santos et al., 2010); it also reduces the (UNICAMP), Campinas-SP, Brazil.
intestinal inflammation in rats by inhibiting the neutrophil chemo- The leaves (50 g) were dried under air circulation, at 40 1C, and
taxis and lipid peroxidation, as well as by scavenging free radicals powdered using a knife mill (46 mesh). The powder was stored at
and improving the local antioxidant defenses (Cestari et al., 2011). 8 1C in hermetically sealed glass flasks. Five grams of the powder
The immunomodulatory effect of B. dracunculifolia extracts is were added to 100 mL of ethanol 90% and shaken for 2 h at 170 rpm,
mediated by their ability to modulate the ROS and cytokine produc- 40 1C, in 125 mL Erlenmeyer flasks (Innova 4300 incubator shaker,
tion by murine macrophages (Bachiega et al., 2013; Missima et al., New Brunswick Scientific, Edison, NJ, USA). After cooling to room
2007). temperature (25 1C), the samples were filtered through analytical
B. dracunculifolia provides most of the chemical compounds filter papers. The solid residue was submitted to a new extraction
found in BGP produced in the states of São Paulo and Minas Gerais, with ethanol, as described above. The filtered extracts were concen-
like flavonoids and cinnamic acid derivatives (Alencar et al., 2005; trated under vacuum, dried and stored at 8 1C until use.
Sousa et al., 2011). Such compounds seem to be responsible for the
biological activities shared by B. dracunculifolia and BGP, like 2.3. Characterization of BdE
antioxidant, anti-carciogenic, anti-inflammatory and immunomo-
dulation (Leitão et al., 2004; Santos et al., 2010; Guimarães et al., 2.3.1. Quantification of total phenolic compounds
2012). Thus, this plant is a promising candidate for the develop- The total phenolic content of the BdE samples was determined by
ment of an effective phytotherapic drug with pharmacological the Folin–Ciocalteau method (Singleton and Rossi, 1965). Concentra-
properties comparable to propolis. Furthermore, B. dracunculifolia tion of these compounds was determined by comparison to the
can be easily cultivated to supply the raw material for phytochem- standard curve. Results are expressed as μM of gallic acid equivalents.
ical and pharmacological studies (Sousa et al., 2011).
However, seasonality and other factors like the interaction with 2.3.2. Quantification of total flavonoids
insects also affect the production of plant secondary metabolites The total amount of flavonoids was quantified as described by
(Gobbo-Neto and Lopes, 2007). Simões-Ambrósio et al. (2010) Costa (1982). Briefly, an aliquot of the extract was added to a medium
reported that seasonality influences the chemical profile of BGP containing 60 mL of glacial acetic acid, 1.0 mL of pyridine:H2O:AlCl3
and its ability to modulate the ROS production by neutrophils. 12% solution (17:80:3) and 1.24 mL of DMSO:H2O (1:1). After 5 min of
The content of phenolic compounds in cultivated B. dracunculifolia incubation at 25 1C, the reaction product was quantified by absor-
varies throughout the year (Sousa et al., 2011), but the impact bance readings at 420 nm. The flavonoid content was determined by
A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664 657

comparison to the standard curve and expressed as mM of quercetin control) – and incubated for 3 min, at 37 1C. The reaction was
equivalents. initiated by adding OZ (1 mg mL  1). The luminol- or lucigenin-
enhanced chemiluminescence (CL-lum and CL-luc, respectively)
2.3.3. HPLC analysis was measured for 15 min, at 37 1C, in a luminometer (Auto Lumat
BdE samples (20 mg) were dissolved in 5 mL of methanol in LB 953, EG&G Berthold, Bad Wildbad, Germany). The light emis-
volumetric flasks, sonicated for 10 min and diluted to 10 mL in sion was recorded in cpm (photons counts per minute).
deionized water containing gallic acid as the internal standard. The The percentage of CL inhibition by each sample was calculated
samples were filtered through a 0.45 μm filter before analysis. using the formula: [1 (IACsample/IACcontrol)]  100, where IAC is the
The BdE samples were analyzed by HPLC using a Shimadzu apparatus integrated area of CL. IC50 values (concentration that inhibits 50% of
equipped with Shimadzu Shim-Pack CLC-ODS column (4.6 mm  the neutrophil CL) were determined by non-linear regression.
250 mm, particle diameter of 5 μm); SPD-M 20A diode-array detector
set at 275 nm; LC-20AT quaternary pump; CBM-20A controller; SIL-
20A autosampler; Shimadzu LC solution software (version 1.21 SP1).
The mobile phase used was aqueous solution of formic acid (0.1% v/v,
pH 2.7) (A) and methanol (B), under a flow rate of 0.8 mL min  1.
An aliquot (10 μL) of each sample was injected. First, a linear gradient
of 20–95% of B was used over a period of 70 min, followed by another
linear gradient of 95–20% of B until 75 min; then the elution was kept
constant until 77 min.
The analytical curves of the standards were prepared and
analyzed in triplicate as described above. The total peak area
was used to determine the correlation coefficients and linear
regression equations. These equations were used to calculate the
concentration of the internal standard, caffeic acid, p-coumaric
acid, artepillin C, AME and isosakuranetin, found in the twelve BdE
extracts analyzed. The results are reported as mean 7standard
deviation.

2.4. Human neutrophils isolation

Twenty adult volunteers in good general health and aged


between 18 and 40 years were recruited according to the protocol
approved by the local Research Ethics Committee on Human
Experimentation (CEP/FCFRP-USP, protocol number 200), which
follows the rules of the 196/96 Resolution of the Brazilian National
Health Council and the Helsinki Declaration of 1975 (revised in
2008). All the participants signed an informed consent prior to the
performance of any study-related procedure. Exclusion criteria
were: (1) history of any acute or chronic disease, (2) recent use of
anti-inflammatory drugs, (3) recent history of alcohol or drug
abuse, or (4) active smoking.
Blood was collected by venous puncture and diluted 1:2 in
Alsever solution (anticoagulant). Neutrophils were isolated by the
gelatin method, as described by Paula et al. (2009). The cell pellets
were suspended in Hanks balanced saline solution (HBSS) contain-
ing 1 mg mL  1 of gelatin (HBSS-gel). The cell preparations con-
tained 90–95% neutrophils with viability higher than 95%, as
established by exclusion with Trypan Blue.

2.5. Preparation of serum-opsonized zymosan (OZ)

Zymosan particles were opsonized with a pool of normal


human serum obtained from at least ten healthy donors, according
to the procedure described by Kanashiro et al. (2004). The serum-
opsonized zymosan (OZ) was suspended in HBSS-gel for use.

2.6. Chemiluminescence assay Fig. 1. Inhibitory effect of Baccharis dracunculifolia extracts (BdE) on the oxidative
metabolism of opsonized zymosan-stimulated human neutrophils, assessed by the
lucigenin- and luminol-enhanced chemiluminescence assays (CL-luc and CL-lum,
The concentration of each component in the final reaction respectively). (A) Representative profile of CL-lum and CL-luc inhibition by different
volume (0.5 mL) is indicated in parentheses. Luminol, lucigenin concentrations of BdE collected in May/07 and quercetin (reference compound).
and BdE stock solutions were prepared in DMSO and diluted in (B) and (C) Inhibition of the CL-luc (B) and CL-lum (C) by BdE obtained from
HBSS-gel prior to use. Aliquots of neutrophils (1  106 cells mL  1) B. dracunculifolia samples collected monthly during one year, expressed as IC50
value (concentration that inhibited the biological response by 50%). Data expressed
were mixed with the chemiluminescence probe (100 mM luminol as the mean 7 SD of five independent experiments performed in duplicate.
or lucigenin) and the sample - BdE (2.5–50 mg mL  1), quercetin Statistics: values not sharing the same letter (a–e) are significantly different from
(0.84–8.45 μg mL  1, reference compound), or DMSO (0.1% v/v, each other (p o0.05; ANOVA and Tukey’s post-hoc test).
658 A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664

2.7. Cytotoxicity evaluation biological activity of the extracts were analyzed by the Spearman
test. po0.05 was considered significant.
The cytotoxic effect of BdE on neutrophils was evaluated as
described by Lucisano-Valim et al., 2002. Values in parentheses
refer to the final concentrations. Briefly, aliquots of neutrophils 3. Results
(1  106 cells mL  1) were incubated with the BdE sample (May/07,
50 μg mL  1), DMSO (0.1% v/v; vehicle control) or Triton X-100 3.1. Seasonality influences the ability of B. dracunculifolia to inhibit
(0.2% v/v; positive control) for 15 min, at 37 1C. The cell pellets the neutrophil ROS generation
were immediately suspended in HBSS-gel after centrifugation
(755g, 10 min, 4 1C). The cellular viability was determined by the We used the lucigenin (CL-luc)- and luminol (CL-lum)-enhanced
Trypan Blue exclusion test, by counting a total of 200 cells for each chemiluminescence assays to evaluate the ability of the B. dracuncu-
sample. The activity of lactate dehydrogenase (LDH) released into lifolia extracts (BdE) to modulate the superoxide anion and total ROS
the supernatant was measured with the LDH Liquiform™ test kit. production by OZ-stimulated human neutrophils, respectively.
We evaluated the effect of BdE prepared from plant leaves harvested
2.8. Data analysis monthly from August/06 to July/07 to assess whether seasonality
influenced the biological activity studied.
Experimental data were processed and analyzed with the aid The twelve BdE inhibited CL-lum and CL-luc in a concentration-
of the GraphPad Prism Software (version 5.00 for Windows, dependent manner (Fig. 1A). For both assays, their IC50 values varied
GraphPad Software Inc., San Diego, CA, USA). Statistical analysis significantly during the studied period; in the neutrophil CL-luc,
was performed by Analysis of Variance (ANOVA) followed by these values ranged from 5.8 to 13.3 mg mL  1 (Fig. 1B). The BdE from
Tukey’s test. Correlation between chemical composition and August/06, September/06 and May/07 inhibited the CL-luc the most

Fig. 2. Quantification of the main chemical markers of the B. dracunculifolia extracts of leaf samples harvested monthly during one year. (A) Total phenolic compounds.
(B) Total flavonoids. (C) The phenolic compounds p-coumaric acid and artepillin C. (D) The flavonoids aromadendrin-4′-methyl ether (AME) and isosakuranetin.
(E) Caffeic acid.
A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664 659

strongly; their IC50 values were 7.671.0, 7.570.9, and


5.871.0 mg mL  1, respectively. The BdE from December/06 and
April/07 inhibited CL-luc least strongly, with IC50 values of
12.771.5 and 13.372.3 mg mL  1, respectively.
The IC50 values obtained for the neutrophil CL-lum inhibition
ranged from 8.1 to 15.8 mg mL  1 (Fig. 1C). The BdE from May/07
(IC50 ¼8.1 71.6 mg mL  1) inhibited the neutrophil CL-lum the
most effectively. The BdE from October/06, December/06 and
January/07 inhibited CL-lum the least effectively; their IC50 values
were 15.6 72.4, 15.3 72.1, and 15.8 72.4 mg mL  1, respectively.
The IC50 values of the BdE from the other months were not
significantly different from each other.
The seasonality plays an important role in the ability of B.
dracunculifolia to inhibit the human neutrophil ROS generation.
Among all the studied extracts, the one collected in May/07
inhibited both CL-luc and CL-lum the most strongly, but they were
less effective than quercetin. The IC50 values of this flavonoid
Fig. 3. Chemical structures of the main compounds identified in the B. dracuncu-
obtained for CL-luc and CL-lum inhibition were 2.05 70.21 and
lifolia extracts. (1) caffeic acid; (2) p-coumaric acid; (3) artepillin C; (4) aromaden-
2.31 70.25 μg mL  1, respectively. drin-4′-methyl ether; (5) isosakuranetin.
Furthermore, the cellular viability and LDH released by the
BdE-treated cells (98.1 70.5% and 5.6 72.1%, respectively) was
similar to the control (98.067 0.3% and 3.1 72.0%, respectively).
Thus, BdE was not toxic to the neutrophils, at least under the but decreased in May/07 and increased in March/07, June/07 and
assessed conditions. July/07.
The analysis of the variation in the concentration of the quantified
compounds observed along the evaluated months suggest that
3.2. Seasonality influences the chemical profile of BdE seasonality influenced the production of AME, isosakuranetin and
artepillin C by B. dracunculifolia more strongly than it influenced the
To study the chemical profile of B. dracunculifolia, we quantified production of caffeic acid and p-coumaric acid.
the concentration of total flavonoids and phenolic compounds,
and analyzed the twelve BdE samples by HPLC.
The total amount of phenolic compounds represented more
than 10% (w/w) of the BdE weight. In general, the concentration of 3.3. Correlation between biological activity and chemical
these classes of compounds did not change significantly through- composition
out the year, ranging from 109.0 73.5 to 172.8 7 10.3 mg g  1
(Fig. 2A). A significant difference (po 0.05) was only detected The correlation between inhibition of superoxide anion and
between the BdE samples collected in August/06 and April/07, the total ROS generation by OZ-stimulated human neutrophils and the
months presenting the lowest and highest levels of phenolic chemical composition of the BdE samples was analyzed by the
compounds. In the other BdE samples, the total phenolic com- Spearman test (Figs. 4 and 5). We used the IC50 values obtained for
pounds were detected in the concentration range from 132.9 73.5 the CL-lum and CL-luc assays as parameters of biological activity,
to 161.777.8 mg g  1. and the mean concentration values of the total phenols, total
Flavonoids constituted less than 40% (w/w) of the total phe- flavonoids and the five quantified compounds as parameters of
nolic compounds detected in the BdE samples. The highest chemical composition.
concentration of flavonoids occurred in December/06, February/ We found that both CL-lum (p¼0.0034; r2 ¼0.5923) and CL-luc
07 and March/07 (35.8 71.4, 35.8 70.4 and 36.3 71.3 mg g  1, (p¼0.0470; r2 ¼0.3389) inhibition correlated positively with the
respectively); the lowest concentrations were found in August/ concentration of caffeic acid (Figs. 4 and 5C). The CL-lum inhibition
06, September/06 and May/07 (20.6 70.6; 24.17 1.8 and 25.5 7 did not correlate with the concentration of the other compounds
0.9 mg g  1, respectively) (Fig. 2B). These groups were significantly studied (Fig. 4A, B and D–G). In contrast, CL-luc inhibition correlated
different from each other (p o0.05). negatively with the content of total flavonoids (p¼0.0043,
The HPLC qualitative profiles of the BdE samples were quite r2 ¼0.5749), artepillin C (p¼0.0148, r2 ¼ 0.4638), and AME (p¼
similar, but there was a huge variation in the amount of some 0.0032, r2 ¼0.5986) (Fig. 5B, E and F), but did not correlate with
compounds. The chromatographic analysis allowed the identifica- the concentration of total phenolic compounds and p-coumaric acid
tion and quantification of three cinnamic acid derivatives, namely (Fig. 5A and D). There was a trend towards a negative correlation
caffeic acid, p-coumaric acid and artepillin C, as well as two (p¼0.0872) of this biological activity with the concentration of
flavonoids: AME and isosakuranetin (Fig. 2C–E). Their chemical isosakuranetin (Fig. 5G).
structures are represented in Fig. 3. In addition, the BdE sample that suppressed the neutrophil
The BdE sample from May/07 presented the highest concentra- CL-lum and CL-luc the most effectively (May/07), contained the
tion of caffeic acid (Fig. 2E) but the lowest concentrations of the highest ratios of caffeic acid to the total phenols and flavonoids,
other four compounds quantified (Fig. 2C and D). Compared with and to the other isolated compounds (Table 1). The lowest ratios
the other samples, the BdE from March/07 contained the highest occurred in the less active BdE samples, collected from October/06
concentration of p-coumaric acid, AME and artepillin C; they were to April/07. This ratio influenced the neutrophil CL-luc inhibition
respectively two-, four- and sevenfold higher than their concen- more strongly than it influenced the CL-lum inhibition. In general,
tration detected in May/07. The highest concentration of isosakur- the samples from August/06, September/06, June/07 and July/07
anetin was found in February/07; it was threefold higher than that – which suppressed the neutrophil CL-luc very effectively – also
detected in May/07. The concentration of p-coumaric acid did not presented high ratios of caffeic acid to the other components of
change significantly in the samples from August/06 to February/07, the extract (Table 1).
660 A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664

Fig. 4. Correlation analysis between inhibition of the luminol-enhanced chemiluminescence (CL-lum) of neutrophils and the amount of total phenolic compounds (A), total
flavonoids (B) and the main compounds (C–G) isolated from B. dracunculifolia. AME: aromadendrin-4′-methyl ether. The CL-lum assay was used to evaluate the total reactive
oxygen production by human neutrophils stimulated with opsonized zymosan. The inhibitory effect of B. dracunculifolia extracts was represented by their IC50 values. Data
expressed as the mean 7SD of five independent experiments performed in duplicate. Correlations were analyzed by the Spearman test, and p o 0.05 was considered
significant.

4. Discussion mechanism to dampen inflammation and tissue injury mediated


by ROS overproduction by neutrophils that can contribute to the
In the present study we assessed whether seasonality affects the overall in vivo anti-inflammatory effect of B. dracunculifolia
chemical composition of B. dracunculifolia leaf extracts (BdE) and (Cestari et al., 2011; Santos et al., 2010). B. dracunculifolia extracts
their ability to inhibit the ROS generation by OZ-stimulated human also modulate the production of ROS and cytokines by murine
neutrophils. Production of the first radical generated, the superoxide macrophages (Bachiega et al., 2013; Missima et al., 2007).
anion, was specifically measured by the lucigenin-enhanced chemi- The ability of BdE to inhibit both, CL-luc and CL-lum, varied
luminescence (CL-luc) assay. The total ROS generation, in particular significantly during the period studied (August/06 to July/07),
those produced by the MPO system, was measured by the luminol- indicating that seasonal factors such as climate change and rainfall
enhanced chemiluminescence (CL-lum) assay (Van Dyke and affect the biological activity of BdE. The BdE sample from May/07
Castranova, 1987). Further, to understand the influence of the BdE inhibited the neutrophil CL-luc and CL-lum the most effectively, but
chemical composition on the aforementioned biological activities, we this effect is not related to toxicity of the BdE on these cells, at least
analyzed the correlation between this parameter and the concentra- under the assessed conditions. These results agree with a previous
tions of the major components of BdE. work that reported the high inhibitory effect of the Brazilian green
All the tested BdE inhibited the neutrophil CL-luc and CL-lum in propolis collected in May on the ROS production by rabbit neutro-
a concentration-dependent manner. This is an important phils (Simões-Ambrósio et al., 2010). B. dracunculifolia is the main
A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664 661

Fig. 5. Correlation analysis between inhibition of the lucigenin-enhanced chemiluminescence (CL-luc) of neutrophils and the amount of total phenolic compounds (A), total
flavonoids (B) and the main compounds (C–G) isolated from B. dracunculifolia. AME: aromadendrin-4′-methyl ether. The CL-luc assay was used to evaluate the superoxide
anion production by human neutrophils stimulated with opsonized zymosan. The inhibitory effect of B. dracunculifolia extracts was represented by their IC50 values. Data
were expressed as the mean7 SD of five independent experiments performed in duplicate. Correlations were analyzed by the Spearman test, and p o 0.05 was considered
significant.

Table 1
Ratio of caffeic acid to the total phenols, total flavonoids, and four compounds isolated from Baccharis dracunculifolia leaf extract.

Sample Total phenols Total flavonoids p-Coumaric acid AME Isosakuranetin Artepillin C

Aug/06 1.0 5.2 14.6 21.4 37.7 24.5


Sep/06 1.0 6.4 20.1 36.1 47.7 49.4
Oct/06 0.9 3.6 16.2 17.7 28.3 26.1
Nov/06 0.9 4.1 17.2 16.7 19.9 33.2
Dec/06 0.9 3.4 15.9 14.6 35.9 18.6
Jan/07 0.9 4.0 18.0 17.8 20.0 32.1
Feb/07 0.8 3.2 14.7 12.2 15.4 22.6
Mar/07 0.9 3.8 14.4 12.1 32.9 17.1
Apr/07 0.8 4.6 18.1 17.5 32.6 19.7
May/07 1.5 8.5 36.9 78.4 94.0 174.4
Jun/07 1.7 7.7 27.7 51.2 79.4 64.9
Jul/07 1.0 4.9 17.4 29.1 42.1 22.4

Data are expressed as (mean concentration of caffeic acid / mean concentration of each compound)  100.
662 A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664

botanical source of this kind of propolis (Alencar et al., 2005), and peroxidases (Andrade et al., 2013; Kato et al., 2003; Moreira
their chemical profiles are qualitatively similar (Park et al., 2002). It et al., 2007). Caffeic acid inhibits the MPO activity more effectively
highlights the importance of the secondary metabolites of both, B. than p-coumaric acid (Kato et al., 2003), and significantly inhibits
dracunculifolia and green propolis, to the antioxidant activity assessed the neutrophil CL-lum and horseradish peroxidase activity (Grael
in cellular experimental models of ROS generation. et al., 2010). AME and isosakuranetin slightly inhibit the rabbit
The twelve BdE samples analyzed were rich in phenolic com- neutrophil CL-lum (Simões et al., 2004).
pounds and flavonoids. The major compounds identified were the Interestingly, the neutrophil CL-luc inhibition by BdE is posi-
flavonoids isosakuranetin and aromadendrin-4′-methyl ether (AME), tively correlated with the caffeic acid concentration, but negatively
and the cinnamic acid derivatives caffeic acid, p-coumaric acid, and correlated with the concentration of total flavonoids, artepillin C,
artepillin C. These results agree with previous reports on the AME and isosakuranetin. In addition, the BdE samples from
literature (Sousa et al., 2011; Leitão et al., 2004; Park et al., 2002). October/06 to April/07, which had the lowest inhibitory effects
The qualitative chemical profiles of the twelve samples were similar, on the neutrophil CL-luc, also had low ratios of caffeic acid to
but the relative amounts of these five compounds varied significantly isosakuranetin, AME and artepillin C. It suggests that these
along the year; the peak of production of most compounds occurred phenolic compounds improve the superoxide production by neu-
in February and March. This result agrees with those reported by trophils or impair the action of caffeic acid and other bioactive
Sousa et al. (2011): the period between December and April are the compounds present in BdE in the intracellular targets of the
best months for the production of phenolic compounds by cultivated activated neutrophils. Elucidating these mechanisms needs further
B. dracunculifolia. Thus, seasonality influences the quantitative more investigation and is out of the scope of this study. Caffeic acid may
than the qualitative production of secondary metabolites by B. have greater beneficial antioxidant effects in the intracellular
dracunculifolia. environment than the other components of Brazilian propolis
Next, we discuss the impact of the variation in the chemical (Nakajima et al., 2009). It is one of the major components of B.
composition of B. dracunculifolia on its biological activity, as well as dracunculifolia responsible for modulating the cytokine production
the relative contribution of the isolated compounds to the overall by murine macrophages, through the inhibition of the transcrip-
antioxidant effect of the extract. The antioxidant activity of plant tion factor NF-κB (Bachiega et al., 2013).
extracts, assessed in cell-free systems, usually correlates positively The structural features of the isolated compounds may explain,
with the total amount of phenolic compounds (Song et al., 2010). at least in part, the results obtained in the CL-luc assay. The
These compounds are responsible for the DPPH reducing ability of flavonoids AME and isosakuranetin lack the 2,3-double bond
B. dracunculifolia (Guimarães et al., 2012) and B. trimera (Oliveira conjugated with the 4-keto group and the 3-hydroxyl group on
et al., 2012). In the present study, we used the OZ-stimulated the C-ring, which is one important structural requirement to inhibit
neutrophils as a cellular model of ROS generation to evaluate the the neutrophil superoxide anion generation (Grael et al., 2010;
antioxidant activity of BdE. Interestingly, we found that this Kanashiro et al., 2004; Moreira et al., 2007). Simões et al. (2004)
biological effect does not correlate significantly with the concentra- reported that these compounds were not able to inhibit the rabbit
tion of total phenolic compounds. The total concentration of neutrophil CL-luc. The prenyl groups of artepillin C increase its
flavonoids was negatively correlated with the inhibition of super- affinity for membranes and improve its antioxidant activity on the
oxide anion production (assessed by CL-luc) but not correlated with membrane lipid peroxidation of Caco-2 cells (Shimizu et al., 2004).
the total ROS generation (assessed by CL-lum) by neutrophils. This interaction can interfere in the assembly and activity of the
Therefore, the inhibitory potency of BdE on the neutrophil oxidative NADPH oxidase complex, and affect the superoxide anion produc-
metabolism depends more on the structural features of the com- tion by the neutrophils.
pounds present in the extract than on their quantity. This hypoth- Scavenging the superoxide anion can also mediate the inhibition
esis is supported by the fact that the amount of total phenolic of the neutrophil CL-luc. The catechol group improves the antiox-
compounds produced by B. dracunculifolia did not change signifi- idant effect and the inhibitory potency of coumarins on superoxide
cantly along the year, but the concentration of some specific anion generation by rat neutrophils (Wu et al., 2009). The presence
phenolic compounds varied significantly, as discussed below. of the catechol group of caffeic acid favors its free radical scavenging
The BdE sample from May/07, which inhibited the neutrophil and metal chelating properties (Gulcin, 2006). The antioxidant
CL-lum and CL-luc the most strongly, contained the lowest con- effect of this compound against H2O2 and superoxide anion is 4–6
centration of all the compounds analyzed, except caffeic acid. The times stronger than ascorbic acid (Nakajima et al., 2009). Caffeic
inhibition of CL-lum correlates positively with the concentration of acid is also a good intracellular antioxidant: it scavenges H2O2,
caffeic acid, but do not correlate with the concentration of the other superoxide anion and hydroxyl radicals in retinal ganglion cells, and
compounds analyzed. The peak of caffeic acid production was improves the antioxidant status of blood lymphocytes (Nakajima
observed in May and June/07; the concentration of this compound et al., 2009; Prasad et al., 2009). In addition, caffeic acid displays a
was similar in these months. However, the concentrations of significant in vivo antioxidant activity by protecting the rat liver
artepillin C, p-coumaric acid, AME and isosakuranetin found in against oxidant-induced injury (Jayanthi and Subash, 2010).
the BdE samples from May/07 were lower than those found in June/ The concentration of p-coumaric acid significantly decreased in
07. The BdE sample from May/07 inhibited the neutrophil CL-lum the sample from May/07, which had the highest inhibitory effect on
more effectively than the sample from June/07. Together, these data the neutrophil ROS generation; however, it remained almost con-
suggest that a high ratio of caffeic acid to the other phenolic stant in the other months and seems not to contribute to the
compounds favors the neutrophil CL-lum inhibition by BdE. variation in this biological activity of B.dracunculifolia. p-Coumaric
The CL-lum generation is widely dependent on the luminol acid has a low intracellular bioavailability (Shimizu et al., 2004) and
oxidation by the neutrophil MPO; the activity of this enzyme is is less effective than caffeic acid as free radical scavenger and
strongly inhibited by catecholic compounds (Andrade et al., 2013; inhibitor of lipid peroxidation (Gulcin, 2006). On the other hand, p-
Kato et al., 2003). Caffeic acid is the only one among the isolated coumaric acid is a good anti-inflammatory and antioxidant com-
phenolic compounds that bears the catechol group. Artepillin pound in vivo: it decreases the joint edema and inflammation, as
C and p-coumaric acid bear one phenolic hydroxyl group, whereas well as improves the tissue and plasma antioxidant status in
the flavonoids AME and isosakuranetin bear a non-vicinal phenolic arthritic rats (Pragasam et al., 2013).
dihydroxyl group. These substituent groups contribute less than In the present study, the correlation analyses discussed above
the catechol group to inhibit the activity of MPO and other were based on the concentration of the major compounds, since
A.S.G. Figueiredo-Rinhel et al. / Journal of Ethnopharmacology 150 (2013) 655–664 663

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Conflict of interest of bee products based on assays of antioxidant capacities. BMC Complementary
and Alternative Medicine 9, 4, http://dx.doi.org/10.1186/1472-6882-9-4.
Németh, T., Mócsai, A., 2012. The role of neutrophils in autoimmune diseases.
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Phenolic enriched extract of Baccharis trimera presents anti-inflammatory and
antioxidant activities. Molecules 17, 1113–1123.
Acknowledgments Park, Y.K., Alencar, S.M., Aguiar, C.L., 2002. Botanical origin and chemical composi-
tion of Brazilian propolis. Journal of Agricultural and Food Chemistry 50,
2502–2506.
The authors are grateful to Mr. Alcides Silva Pereira and Mrs. Paula, F.S., Kabeya, L.M., Kanashiro, A., Figueiredo, A.S.G., Azzolini, A.E.C.S., Uyemura,
Nadir Mazzucato (Faculdade de Ciências Farmacêuticas de Ribeirão S.A., Lucisano-Valim, Y.M., 2009. Modulation of human neutrophil oxidative
Preto, Universidade de São Paulo, Brazil) for their helpful technical metabolism and degranulation by extract of Tamarindus indica L. fruit pulp. Food
and Chemical Toxicology 47, 163–170.
assistance, and to the Brazilian agencies Fundação de Amparo à Pragasam, S.J., Venkatesan, V., Rasool, M., 2013. Immunomodulatory and anti-
Pesquisa do Estado de São Paulo (FAPESP, Grant # 2004/01962-1) inflammatory effect of p-coumaric acid, a common dietary polyphenol on
and Conselho Nacional de Desenvolvimento Científico e Tecnoló- experimental inflammation in rats. Inflammation 36, 169–176.
Prasad, N.R., Jeyanthimala, K., Ramachandran, S., 2009. Caffeic acid modulates
gico (CNPq, Grant # 473657/2007-4) for financial support.
ultraviolet radiation-B induced oxidative damage in human blood lymphocytes.
Journal of Photochemistry and Photobiology B: Biology 95, 196–203.
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