Lycopene rich extract from red guava (
Psidium guajava
L.) displays
anti-in
fl
ammatory and antioxidant pro
fi
le by reducing suggestive
hallmarks of acute in
fl
ammatory response in mice
Andreanne G. Vasconcelos
a, Adriany das G.N. Amorim
a, Raimunda C. dos Santos
a, Jessica Maria T. Souza
a,
Luan Kelves M. de Souza
b, Thiago de S.L. Araújo
b, Lucas Antonio D. Nicolau
c, Lucas de Lima Carvalho
c,
Pedro Everson A. de Aquino
d, Conceição da Silva Martins
e, Cristina D. Ropke
f, Pedro Marcos G. Soares
c,
Selma Aparecida S. Kuckelhaus
g, Jand-Venes R. Medeiros
b, José Roberto de S.A. Leite
a,g,⁎
aNúcleo de Pesquisa em Biodiversidade e Biotecnologia-BIOTEC, Universidade Federal do Piauí-UFPI, Campus Ministro Reis Velloso-CMRV, Parnaíba, PI, Brazil bLaboratório de Fisio-Farmacologia Experimental-LAFFEX, Universidade Federal do Piauí-UFPI, Campus Ministro Reis Velloso-CMRV, Parnaíba, PI, Brazil cLaboratório de Farmacologia da Inflamação e do Câncer-LAFICA, Universidade Federal do Ceará-UFC, Fortaleza, CE, Brazil
dLaboratório de Neurofarmacologia, Universidade Federal do Ceará-UFC, Fortaleza, CE, Brazil
eNúcleo de Estudos em Microscopia e Processamento de Imagens-NEMPI, Universidade Federal do Ceará-UFC, Fortaleza, CE, Brazil fPhytobios Nordeste LTDA, Parnaíba, Piauí, Brazil
gÁrea de Morfologia da Faculdade de Medicina, Universidade de Brasília-UnB, Brasília, DF, Brazil
a b s t r a c t
a r t i c l e
i n f o
Article history:
Received 30 October 2016
Received in revised form 4 January 2017 Accepted 20 January 2017
Available online 21 January 2017
This study investigated the anti-inflammatory activity of the extract (LEG) and purified (LPG) lycopene from guava (Psidium guajavaL.), as well as some mechanisms possibly involved in this effect. The anti-inflammatory activity was initially assessed using paw edema induced by Carrageenan, Dextran, Compound 48/80, Histamine and Prostaglandin E2 inSwissmice. A peritonitis model was used to evaluate neutrophil migration, the activity of myeloperoxidase (MPO) and reduced glutathione (GSH) concentration; while the effect on the expression of iNOS, COX-2 and NF-κB, was assessed by immunohistochemistry analysis. Results showed that oral and intraper-itoneal administration of LEG and LPG inhibited inflammation caused by carrageenan. LPG (12.5 mg/kg p.o.) sig-nificantly inhibited the edema formation induced by different phlogistic agents and immunostaining for iNOS, COX-2 and NF-κB. Leukocytes migration in paw tissue and peritoneal cavity was reduced, as well as MPO concen-tration, whereas GSH levels increased. Thus, lycopene-rich extract from red guava has beneficial effect on acute inflammation, offering protection against the consequences of oxidative stress by downregulating inflammatory mediators and inhibiting gene expression involved in inflammation.
© 2017 Elsevier Ltd. All rights reserved.
Keywords:
Carotenoids Natural antioxidant
Psidium guajavaL. fruit Inflammation
1. Introduction
Inflammation consists of an answer of the organism, triggered by different types of injuries on the tissues or for infectious agents. It is characterized by increasing vascular permeability, cellular migration and the release of cytokines and free radicals (Medzhitov, 2008; Nourshargh & Alon, 2014). Although inflammatory answer is
consid-ered a protective event, it represents an aggression to the organism, once it results in tissue damage, edema and pain (Medzhitov, 2008). Furthermore, pro-inflammatory mechanisms may contribute to the
de-velopment of chronic diseases, such as diabetes, cancer, arthritis,
neurological diseases and psoriasis, that is why the control of the
in-flammatory process is desired (Lee et al., 2013; Pawelec, Goldeck, & Derhovanessian, 2014; Haworth & Buckley, 2015; Kim, Na, Myint, & Leonard, 2015; Grine, Dejager, Libert, & Vandenbroucke, 2015).
Many researches have focused on new bioactive molecules, natural products and functional foods as alternatives to the development of new anti-inflammatory agents (Moro et al., 2012; Pereira et al., 2012;
Cavalcanti et al., 2013; Pérez et al., 2014). Some food compounds such as antioxidants, singly or in association, might influence the infl amma-tory process, reducing its harmful effects and the risk to develop dis-eases (Stoner & Wang, 2013; Lu & Yen, 2015; Nidhi, Sharavana, Ramaprasad, & Vallikannan, 2015).
Studies show that lycopene, or fractions rich in lycopene, has an im-portant anti-inflammatory behaviour (Renju & Muraleedhara Kurup, 2013; Kim, Park, Kim, & Cho, 2014; Li, Deng, Liu, Loewen, & Tsao, 2014). Lycopene is a carotenoid composed by an acyclic chain with 11 ⁎ Corresponding author at: Área de Morfologia da Faculdade de Medicina (AM),
Faculdade de Medicina (FM), Universidade de Brasília (UnB),CampusUniversitário Darcy Ribeiro, Asa Norte, Brasília, Distrito Federal, DF 70910900, Brazil.
E-mail addresses:jrsaleite@gmail.com,jrleite@pq.cnpq.br(J.R.S.A. Leite).
http://dx.doi.org/10.1016/j.foodres.2017.01.017 0963-9969/© 2017 Elsevier Ltd. All rights reserved.
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Food Research International
conjugated double bonds, especially found in theall-transconfiguration, but also present in a wide variety of cisisomers (Bramley, 2000; Srivastava & Srivastava, 2015). It is recognized as a powerful antioxi-dant, considered to be the most efficient singlet oxygen scavenger among carotenoids, and it is usually found in red or orange fruits and vegetables, among them, red guava (Di Mascio, Raiser, & Sies, 1989; Nimse & Pal, 2015; Nwaichi, Chuku, & Oyibo, 2015).
Guava (Psidium guajavaL.) is a typical fruit of tropical and subtropi-cal regions, popularly used as food and medicine. It has high nutritional value, as well as high antioxidant potential, and shows important anti-hypertensive, antispasmodic, antimicrobial, hypoglycaemic, anodyne, and anti-inflammatory properties (Gutierrez, Mitchell, & Solis, 2008;
Araújo et al., 2014; Flores, Wu, Negrin, & Kennelly, 2015). This study supports, for thefirst time, the anti-inflammatory, antioxidant effects and some partial mechanisms of lycopene fractions obtained from red guava (Psidium guajavaL.), generating information that allows biotech-nological applications for the fruit and for the benefit of human health.
2. Materials and methods
2.1. Chemicals
Indomethacin,λ-Carrageenan, o-dianisidine dihydrochloride, di-methyl sulfoxide (DMSO) and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), Dextran, Compound 48/80 (C48/80), Histamine and Prosta-glandin E2 (PGE2) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Heparin and hydrogen peroxide (H2O2) were pur-chased from Merck (Darmstadt, Germany). All samples were prepared in 10% DMSO. Dichloromethane, Chloroform and Ethanol were used for the extraction and purification process of lycopene.
2.2. Animals
Male and female Swiss mice (30 ± 5 g) were maintained at temper-ature control (24 ± 2 °C) under a 12/12 light/dark cycle with free access to water and food. The Ethics Committee in Research of the Federal Uni-versity of Piauí previously approved all experiments, under protocol number 068/14, and the procedures were performed according the Guide for Care and Use of Laboratory Animals(National Institute of Health, Bethesda, MD, USA).
2.3. Extraction and purification of lycopene from guava
Lycopene obtained from red guava (P. guajavaL.) was extracted in accordance with the methodology developed byAmorim, Leite, and Ropke (2016)detailed in the patent n° BR102016030594-2. The extract was obtained from 100 g of fresh guava, using organic solvent (dichlo-romethane), producing LEG (Lycopene Extract from Guava). Subsequently, the extract was stored in solvent overnight under refrig-eration at 4 °C, posteriorly washed with ethanol and dried in concentra-tor plus (Eppendorf, Hamburg, Germany), producing LPG (Lycopene Purified from Guava). All isolation steps were carried out under dim light and, at thefinal step of the process, under nitrogen atmosphere. Lycopene extract and purified lycopene were reconstituted in 10%
DMSO and used for biological assays.
2.4. Evaluation of anti-inflammatory activity
2.4.1. Carrageenan-induced paw edema
Carrageenan-induced paw edema was carried out according toSilva et al. (2013)method. Lycopene extract from guava (LEG; 25, 50 or 100 mg/kg) and lycopene purified from guava (LPG; 12.5, 25 or 50 mg/kg) were tested by intraperitoneal (i.p.) or oral (p.o.) rote in an-imals randomly divided into groups (n= 5). The edema was induced by intraplantar (i.pl.) injection of 50 μL of a carrageenan suspension (500μg/paw in 0.9% sterile saline) into the right hind paw. The animals
received a pretreatment with Indomethacin (10 mg/kg, i.p.), as an anti-inflammatory reference drug; LEG and LPG carrier (10% DMSO, i.p. or
p.o.) as negative control; or with the samples, thirty minutes before the edema induction. Paw volume was measured immediately after the stimulus (Vo: basal volume) and 1, 2, 3, and 4 h later (Vt) by water displacement at plethysmometer (LE 7500, Panlab, Barcelona, Spain). The edema percentage inhibition for the treated group, com-pared to the positive control group, which received carrageenan, was calculated by the following formula:
Inhibitionð Þ ¼% ðVt–VoÞControl−ðVt–VoÞTreatment100
Vt–Vo ð ÞControl
2.4.2. Paw edema induced by different inflammatory agents
According to each group (n= 5) specification, the animals were pre-treated with LPG (12.5 mg/kg, p.o.), 10% DMSO (p.o.) or Indomethacin (10 mg/kg, i.p.), as a reference drug. The edema was induced by intraplantar (i.pl.) injection of 50μL of Dextran (500μg/paw), C48/80 (12μg/paw), Histamine (100μg/paw) or Prostaglandin E2 (PGE2, 3 nmol/paw) into the right hind paw, 30 min later. The paw volume of the animals in PGE2, Histamine and C48/80 groups was measured im-mediately after the stimulus (Vo: basal volume) and 30, 60, 90 and 120 min later (Vt) by water displacement at Plethysmometer (LE 7500, Panlab, Barcelona, Spain), while the paw volume of the animals in Dextran group was measured immediately after the stimulus and every hour for 4 h, by the same method reported above.
2.4.3. Histopathological analysis
Aiming to assess the degree of inflammation, the groups submitted to carrageenan-paw edema tests underwent histopathological analysis. The skin obtained from Swiss mice footpad werefixed in 10% buffered formaldehyde for 24 h, dehydrated in increasing concentrations of eth-anol, diaphanized in xylene, impregnated and embedded in paraffin, sectioned (5μm) and stained with Gomori's Trichrome. Images of histo-logical sections were captured by the Microscope Primo Star® (Carl Zeiss, Jurubatuba, São Paulo, Brazil) with 1000× of magnification. The histopathological analyses were carried out by one observer, in all histo-logical sections, to evaluate the architecture of the tissues and into two equidistant areas of the histological images (175μm2), to quantify the number of inflammatory cells. The results were expressed as mean ±
standard deviation.
2.4.4. Immunohistochemistry analysis for iNOS, COX-2 and NF-kB on edem-atous paw sections from carrageenan- induced edema in LPG-treated mice Immunohistochemistry assays were performed by streptavidin-bio-tin-peroxidase method (Hsu, Raine, & Fanger, 1981). The animals were treated in accordance with the specification of each group (n= 5), with
LPG (12.5 mg/kg, p.o.), 10% DMSO (LPG vehicle, p.o.) or Indomethacin (10 mg/kg, i.p.), 60 min before intraplantar injection of carrageenan (500μg/paw in 0.9% sterile saline). The animals were sacrificed 3 h
later and 5 mm sections of the plantar region from the carrageenan-injected hind paw were immersed in 10% buffered formol for 48 h to histological processing. Deparaffinized and rehydrated paw sections
(5μm) were immersed in 0.1 M citrate buffer (pH 6) under 18 min mi-crowave heating for antigen recovery.
diaminobenzidine-peroxide (DAB) cromophore, counter-stained with Mayer hematoxylin, dehydrated and mounted in microscope slides for analyses. The data were semiquantified, as relative optic density, with the Image J (NIH, USA) software.
2.4.5. Leukocyte migration assessment by peritonitis model
Leukocyte migration was evaluated by peritonitis model, adminis-trating 250μL of a carrageenan suspension (500μg/cavity, i.p.) 30 min after the pre-treatment with 10% DMSO (LPG vehicle, p.o.), Indometha-cin (10 mg/kg, i.p.) or LPG (12.5 mg/kg, p.o.). The animals were eutha-nized 4 h later and the peritoneal cavity was washed with 1.5 mL of heparinized phosphate-buffered saline (PBS), to obtain peritoneal cells. Total cell count was performed in Neubauer chamber. The differ-ential 100 cell count was performed in slides prepared using a cytocentrifuge, stained with hematoxylin and eosin, and examined with an optical microscope. Part of the peritoneal exudate obtained was stored under freeze for later biochemical determinations.
2.4.6. Determination of myeloperoxidase (MPO) activity
Myeloperoxidase (MPO) activity was assessed by a colorimetric method, using the peritoneal exudate obtained. The reaction was per-formed in a 96 well-plate. A volume of 10μL of the sample, which was previously centrifuged (3000 rpm, 20 min at 4 °C), was added in the plates, followed by a volume of 200μL of a solution containing o-dianisidine dihydrochloride and 1% of hydrogen peroxide. The results were obtained by measuring the change in absorbance at 450 nm in a one-minute interval. The results were expressed as MPO unit/mL of peritoneal exudate (U/mL), considering that the unit of MPO activity corresponds to the conversion of 1μmol of hydrogen peroxide to water in 1 min at 22 °C.
2.4.7. Reduced glutathione (GSH) levels determination
Glutathione (GSH) levels determination was assayed using perito-neal exudate, according to adaptations from the method previously de-scribed (Sedlak & Lindsay, 1968). The sample was previously vortexed to provide the deproteinization and release of GSH content from the cells. Subsequently, it was centrifuged (3000 rpm, 20 min at 4 °C) and,
finally, 400μL of the supernatant were mixed with Tris buffer (800μL, 0.4 M) and 20μL 0.01 M DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid). The mixture was stirred for 3 min and read at 412 nm in spectropho-tometer (Shimadzu, Kyoto, Japan). Glutathione concentration was cal-culated based on GSH standard curve. The results were expressed as micrograms of GSH/mL of peritoneal exudate (μg/mL).
2.5. Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA). Newman-Keuls multiple comparisons post-test was used to determinate the statistical significance of differences among groups in the paw edema and biochemical experiments, as well as Tukey test was used for immunohistochemistry analysis. Considering the paramet-ric data,t-Test was used to compare histopathological data obtained for two independent groups. Analyses were performed employing PRISM® 5.0 software package (GraphPad, USA, 2005). Results are expressed as means ± SEM, andpb0.05 was set as statistical significance.
3. Results
3.1. Anti-inflammatory effect of lycopene fractions from guava in carra-geenan-induced paw edema
The edema formation was effectively induced by the administration of carrageenan (500μg/paw), reaching its peak 3 h after injection (0.061 ± 0.007 mL) (Figs. 1 and 2). Lycopene extract from guava (LEG; 25, 50 or 100 mg/kg) and lycopene purified from guava (LPG;
12.5, 25 or 50 mg/kg) were administrated through oral and intraperito-neal routes. LEG (50 and 100 mg/kg) and LPG (12.5 and 25 mg/kg) sig-nificantly inhibited (p b 0.05) the edema formation, by both
administration routes, similarly to the effect obtained by the reference drug Indomethacin (10 mg/kg, i.p.), at the same level, at the third hour. Among them, the maximum inhibitory effect was observed with LEG 50 mg/kg i.p. (0.012 ± 0.006 mL) (Fig. 1A), LEG 100 mg/kg p.o. (0.012 ± 0.004 mL) (Fig. 1B) and LPG 12.5 mg/kg p.o. (0.018 ± 0.006 mL) (Fig. 2B), presenting 80.33%, 80.33%, 70.49% of inhibition, re-spectively. LPG 12.5 mg/kg p.o. reaches 96.72% inhibition (0.002 ± 0.002 mL) in the fourth hour. In contrast, no statistically significant anti-edematogenic effect was observed in LEG 25 mg/kg i.p. or p.o. and LPG 50 mg/kg i.p groups. Furthermore, in the group treated with LPG 50 mg/kg p.o. there was an edema formation greater than (pb0.05) carrageenan only. Therefore, considering lower dose, high
de-gree of purity and the route of administration convenience, LPG 12.5 mg/kg p.o. was selected for the subsequent analysis of the
anti-in-flammatory effect observed.
3.2. Anti-inflammatory effect of LPG in paw edema induced by different agents
Paw edema formation induced by Dextran, C48/80, Histamine and PGE2 reaches its peak with 2 h (0.056 ± 0.010 mL), 30 (0.070 ±
0.012 mL), 60 (0.072 ± 0.008 mL) and 60 (0.045 ± 0.005 mL) minutes after the administration of the phlogistic agent, respectively (Fig. 3). The pre-treatment with LPG (12.5 mg/kg, p.o.) inhibited significantly (pb0.05) the paw edema formation peak, presenting 77.68% (Dextran),
57.14% (C48/80), 65.27% (Histamine) and 82.22% (PGE2) of inhibition, in the above times. On the other hand, the pre-treatment with Indo-methacin (10 mg/kg, i.p.) inhibited significantly (pb0.05) the paw
edema formation peak induced by Dextran and Histamine, presenting 95.54% and 61.11% of inhibition, respectively, but had no significant ef-fect on C48/80 and PGE2. Comparing LPG and Indomethacin groups, no statistically significant difference was observed in the inhibition of the
paw edema formation induced by Dextran and Histamine.
3.3. Histopathological analysis
Histopathological analyzes showed that the tissues, epithelial and connective, were preserved in the skin of the animals of the
different groups submitted to carrageenan-paw edema tests (Fig. 4). However, the number of migrating cells in the animals treat-ed with Indomethacin (10 mg/kg i.p. = 5.2 ± 1.5 cells/175μm2), Car-rageenan (500 μg/paw i.pl. = 9.4 ± 3.1 cells/175 μm2), LPG (12.5 mg/kg p.o. = 5.84 ± 2.1 cells/175μm2; 25 mg/kg p.o. = 4.8 ± 1.2 cells/175μm2; 50 mg/kg i.p. = 9.9 ± 3.4 cells/175μm2) or LEG (25 mg/kg i.p. = 7.2 ± 0.8 cells/175μm2; 50 mg/kg i.p. = 5.0 ± 0.7 cells/175 μm2; 100 mg/kg p.o. = 10.4 ± 2.1 cells/ 175μm2) was higher in the reticular dermis when compared to the 10% DMSO vehicle control group (0.2 ± 0.2 cells/175μm2) (Testt, pb0.001). The results also showed intense neutrophilic infiltration
in the skin of animals, induced by carrageenan (500μg/paw), when compared (Testt,pb0.05) to those animals previously treated
with Indomethacin (10 mg/kg i.p.), LPG (12.5 or 25.0 mg/kg p.o.) or LEG (50.0 mg/kg i.p.) (Fig. 5).
1.1. 3.4. LPG decreases iNOS, COX-2 and NF-κB immunoexpression on edematous paw sections
Fig. 2.Effect of Lycopene Purified from Guava (LPG 12.5, 25 or 50 mg/kg), administered by intraperitoneal (A) and oral (B) routes, on the formation of carrageenan-induced paw edema, 1, 2, 3 and 4 h after the stimulus with the phlogistic agent. Indomethacin (10 mg/kg, i.p.) was used as a reference drug. The values were expressed as mean ± SEM. *pb0.05vsCg + 10% DMSO group; **pb0.05vs10% DMSO. Cg = carrageenan; Indo = Indomethacin; p.o. =per os(by mouth); i.p. = intraperitoneal.
Immunohistochemical assay showed that immunostaining for iNOS (Fig. 6B), COX-2 (Fig. 6F) and NF-κB (Fig. 6J) in some cells in the paw conjunctive tissue from animals that received carrageenan was signifi -cantly (pb0.05) intense (90.40 ± 11.92, 67.20 ± 7.59 and 72.75 ±
11.31 immunostained cells/5fields, respectively) when compared
with 10% DMSO negative control group (13.00 ± 6.79, 8.20 ± 1.65 and 20.00 ± 3.19 immunostained cells/5fields, respectively). The pre-treatment with LPG (12.5 mg/kg, p.o.) significantly (pb0.05) reduced
the immunostaing for iNOS (Fig. 6C), COX-2 (Fig. 6G) and NF-κB (Fig.
6L) compared with the carrageenan group, presenting 26.60 ± 4.01, 34.40 ± 8.11 and 26.20 ± 5.84 immunostained cells/5fields, respective-ly. Additionally, the animals that received indomethacin, the reference drug, also showed significant immunostaining reduction for iNOS (Fig. 6D), COX-2 (Fig. 6H) and NF-κB (Fig. 6M) (61.20 ± 11.09, 39.60 ±
5.92 and 31.80 ± 14.25 immunostained cells/5fields, respectively) when compared with the group that received only carrageenan.
3.4. LPG inhibited neutrophil migration at carrageenan-induced peritonitis
The evaluation of neutrophil migration was conducted in peritonitis model, by oral treatment with 10% DMSO (p.o.), Indomethacin (10 mg/kg, i.p.) or LPG (12.5 mg/kg, p.o.). Carrageenan administration induced significantly increase (pb0.05) in total leukocytes (11.40 ±
1.77 × 103 cells/mL, Fig. 7A), with great neutrophil recruitment (7.00 ± 0.46 × 103cells/mL,Fig. 7B). In contrast, pre-treatment with LPG (12.5 mg/kg, p.o.) significantly reduced the leukocyte migration
indomethacin effect (10 mg/kg, i.p.) (6.64 ± 1.30 × 103cells/mL and 1.82 ± 0.40 × 103cells/mL, respectively).
3.5. LPG inhibited the increase of MPO activity
The MPO activity assessment in peritoneal exudate showed signifi -cantly (pb0.05) increased levels in the carrageenan group (199.90 ±
38.67 U/mL,Fig. 8). The pre-treatment with LPG (12.5 mg/kg, p.o.) inhibited (58.26 ± 10.24 U/mL) the increase of MPO activity caused by carrageenan. This result was similar to the indomethacin effect (49.65 ± 11.7 U/mL).
3.6. LPG induced the increase of reduced GSH levels
Fig. 9shows that LPG (12.5 mg/kg, p.o.) significantly increased the GSH levels (396.00 ± 31.07μg/mL), while carrageenan reduced them (88.60 ± 15.72μg/mL). Indomethacin (10 mg/kg, i.p.), the reference drug, maintained GSH concentration to basal levels (211.00 ± 59.52μg/mL and 242.63 ± 45.26μg/mL, respectively). The LPG and In-domethacin groups were significantly (pb0.05) different.
4. Discussion
Guava is a fruit rich in antioxidant composites, such as phenolic com-pounds, anthocyanins,flavonoids, triterpenes, ascorbic acid and carot-enoids (Flores et al., 2015; Nwaichi et al., 2015). Extractions with organic solvents showed that the highest content of carotenoids present in the composition of extracts of pink guava are of lycopene and beta-carotene (Chandrika, Fernando, & Ranaweera, 2009; Kong et al., 2010). The extraction and purification methodology with organic solvent, developed byAmorim et al. (2016), which was employed in this study, reveals lycopene-rich extract from red guava contains until 40% of lycopene/extract dry weight and the purified lycopene concentrate contains from 40% to 90% of lycopene/extract dry weight.
These compounds have been related to several beneficial effects for human health and served as the basis for the development of new ther-apeutic alternatives (Renju, Muraleedhara Kurup, & Saritha Kumari, 2013; Lu & Yen, 2015). Previous studies have shown that the extracts obtained from the leafs or fruits from guava tree (Psidium friedrichsthalianum) exhibit anti-inflammatory activity associated to
the presence of phenolic compounds, by inhibition of pro-inflammatory mediators and reduction of leukocytes migration (Flores et al., 2013; Jang et al., 2014; Araújo et al., 2014).
The present study shows the anti-inflammatory effect of lycopene extract and lycopene purified from red guava (Psidium guajavaL.). Al-though the anti-inflammatory activity of lycopene from different Fig. 5.Total neutrophils in the reticular dermis of Swiss mice footpad. The cells were
quantified in an area of 175μm2of the histological sections obtained from animals of the 10% DMSO, Carrageenan + 10% DMSO (Cg), Carrageenan + Indo (Indo), Carrageenan + LPG or LEG. The values were expressed as mean ± SD. *pb0.05vs carrageenan group; **pb0.05vs10% DMSO group.
sources is well established, this is thefirst proposal about the use of guava, a fruit popularly used for food and medical purposes, as a source of lycopene with anti-inflammatory potential.
The anti-inflammatory effect was initially demonstrated using carra-geenan-induced paw edema model, which is a model of local acute
in-flammation, considered one of the most widely used and most appropriated methods to study the anti-inflammatory activity of bioac-tive compounds (Posadas et al., 2004; Silva et al., 2015). The paw vol-ume measurement and the histopathological analysis from carrageenan-induced edema in mice showed that LEG and LPG exhibit significant anti-inflammatory activity.
The inflammatory response triggered by carrageenan is a two-phase event: thefirst phase (1−2 h) is characterized by the increase of vascu-lar permeability due to the release of histamine and serotonin by mast cells; the second phase (3–4 h) is marked by neutrophil infiltration and the release of bradykinin, prostaglandin E2 (PGE2), cytokines (IL-1β, TNF-α, IL-10) and NO, as well as free radicals derived from neutro-phils (Vinegar, Schreiber, & Hugo, 1968; Silva et al., 2014; Coura et al., 2015).
LEG and LPG were more effective in the second phase of carrageen-an-induced edema formation, suggesting that the anti-inflammatory
ef-fect observed might be related to the modulation of inflammatory mediators, cytokines release, reduction of neutrophil infiltration and the decrease of oxidative stress.
Regarding the route of administration, no statistically significant dif-ference was observed on the effect of the treatments using oral or intra-peritoneal route. So, to the following studies, with the purpose of investigating some of the mechanisms that might be involved in the anti-edematogenic effect of guava lycopene, LPG 12.5 mg/kg with oral administration was selected, due to its optimal activity with lower
dose, greater convenience of the route of administration and the larger purity grade of the compound.
Furthermore, our data point-out that, regarding to the potency of the anti-inflammatory effect, LPG (12.5 mg/kg; p.o.) was as good as lyco-pene from tomato, reported in literature, which is the most extensively studied and used source of lycopene by pharmaceutical, food and cos-metic industries (Bignotto et al., 2009; Li et al., 2014).Li et al. (2014), studying the anti-inflammatory activity of a tomato extract (Solanum lycopersicumL.), obtained by extraction with ethanol:hexane, adminis-trated orally in Wistar rats, observed significant reduction of the carra-geenan-induced paw edema, with optimum dosage equivalent to 50 mg/kg of lycopene (30 g of extract). Similarly, Bignotto et al. (2009), studying the anti-inflammatory effect of a commercially avail-able suspension containing 10% synthetic lycopene in carrageenan-in-duced paw edema, observed a significant reduction of the edema formation, using dosages of 25 and 50 mg/kg, and treating intraperitoneally.
The evaluation of the mechanisms involved in anti-edematogenic activity was carried out by the administration of different phlogistic agents. Dextran partially induces mast cells degranulation and produces osmotic edema with low protein content and neutrophils (Lo, Almeida, & Beaven, 1982; Coura et al., 2015). Compound 48/80 induces intense release of histamine and serotonin from mast cells by destabilizing the membrane and causing degranulation of these cells, possibly through MRG (Mas-related gene) receptor (Tatemoto et al., 2006; Staats et al., 2013; Silva et al., 2015).
Histamine is an essential mediator in the early events of the infl
am-matory response, which operates mainly in increased vascular perme-ability and vasodilation (Mani, Ramasamy, & Majeed, 2013). PGE2 is also a pro-inflammatory mediator that triggers vascular changes with Fig. 7.Effect of Lycopene Purified from Guava (12.5 mg/kg, p.o.) on inflammatory cells migration from peritoneal exudate. Peritonitis was induced by carrageenan (500μg/cavity, i.p.) administration, 30 min after the pre-treatment with 10% DMSO (p.o.), Indomethacin (10 mg/kg, i.p.) or LPG (12.5 mg/kg, p.o.). Leukocyte migration was evaluated 4 h later. (A) Leukocytes counts and (B) neutrophils counts. The results were expressed as mean ± SEM of 5–6 animals per group. *pb0.05vscarrageenan group; **pb0.05vs10% DMSO-treated group.
Fig. 8.Effect of Lycopene Purified from Guava (12.5 mg/kg, p.o.) on myeloperoxidase (MPO) activity, in peritonitis model. The determination of MPO activity was performed by the reaction of the peritoneal exudate (10μL) with a solution (200μL) containing o-dianisidine and 1% hydrogen peroxide. The change in the absorbance was measured at 450 nm in 1 min interval. The results were expressed as mean ± SEM of 5–6 animals per group. *pb0.05vscarrageenan group; **pb0.05vs10% DMSO-treated group.
other mediators, amplifying the inflammatory response (Kumar, Al-Abbasi, Verma, Mujeeb, & Anwar, 2015). Ourfindings demonstrated
that LPG (12.5 mg/kg; p.o.) was able to inhibit the initial phase of the edema formation induced by Dextran, C48/80 and PGE2, since it had long-lasting inhibitory effect on edema induced by Histamine. Thus, LPG anti-edematogenic effect might involve the stabilization of mast cells cellular membrane and the inhibition of the release and/or action of inflammatory mediators.
Indeed, immunohistochemistry data demonstrated that the anti-edematogenic effect of lycopene purified from red guava might involve the inhibition of the production of pro-inflammatory mediators. Immu-nostaining for COX-2 and iNOS was reduced on the paw tissue from an-imals treated with LPG (12.5 mg/kg p.o.) when compared to carrageenan group. The Cyclooxygenase-2 (COX-2) enzyme corre-sponds to COX inducible isoform, markedly expressed at sites of infl
am-mation, contributing to the production of the pro-inflammatory mediator Prostaglandin (PG), which promotes vasodilatation and is in-volved in the pathogenesis of fever and pain (Abdelazeem et al., 2014). The inducible Nitric Oxide Synthase (iNOS) is an enzyme undetect-able in basal conditions, but its expression can be induced during
in-flammatory answer by cytokines, bacterial lipopolysaccharide and other agents (Förstermann & Sessa, 2012). When induced in macro-phages, iNOS produces large amounts of NO, which promotes vasodila-tion and has cytotoxic effects at high concentravasodila-tions (Förstermann & Sessa, 2012).
Thus, lycopene purified from red guava reduced PGE2 and NO through the inhibition of the expression of enzymes responsible for its production in inflamed tissue, consequently, reducing the inflammatory
effect caused by carrageenan. This data also supports the paw edema in-duced by carrageenan initial test, wherein the anti-edematogenic effect was more effective in the late phase, known to involve the production of PGE2 and NO by leukocytes.
At the same time, LPG-treated group showed low immunopositivity for nuclear factor-kappaB (NF-κB) on the inflamed paw tissue when compared with carrageenan group. This result suggests that - lycopene purified from red guava inhibits inflammatory effects by preventing the expression of genes involved in inflammation, considering that NF-κB, in our study the p65 (also known as RelA), a subunit portion which is found after dissociate the inactive cytosolic complex p65/p50, is a tran-scription factor that plays a critical role in regulating genes expression responsible for inflammation, such as iNOS, COX-2, IL-1βand TNF-α, proliferation and apoptosis (Grossmann, Nakamura, Grumont, & Gerondakis, 1999; Abdelazeem et al., 2014; Kumar et al., 2015).
In other studies, commercial lycopene inhibited the enhancement of NF-kB and COX-2 expression induced by lipopolysaccharide (LPS) in BV-2 microglia, mouse primary cultured microglia and rat primary cul-tured microglia, indicating that it might be useful as a therapeutic agent for neuroinflammation-associated disorders (Lin et al., 2014).
Reinforc-ing ourfindings,Palozza, Catalano, Simone, and Cittadini (2012) em-phasize that lycopene regulates redox molecules as NF-κB and might decrease iNOS and COX-2 gene expression, acting as a non-toxic agent for the control of pro-inflammatory genes.
Cellular migration and the redox-protector effect of LPG (12.5 mg/kg, p.o.) were evaluated. Leukocyte migration, principally neutrophils, to the injured and infected areas plays a key role during the inflammatory process. Circulating neutrophils migrate to the tissues in response to the chemotactic signals released by several cytokines, and can cause tissue injury and the exacerbation of the inflammation process through its phagocytic action (Nourshargh & Alon, 2014).
Literature has revealed some bioactive compounds with anti-infl
am-matory activity that influence the leukocyte migration (Silva et al.,
2013; Silva et al., 2014; Silva et al., 2015). In this study, the histopatho-logical analysis demonstrated that cell recruitment in inflamed paw tis-sue was significantly reduced in mice pretreated with lycopene purified
from red guava. Furthermore, LPG (12.5 mg/kg, p.o.) showed effective-ness in the inhibition of leukocyte migration and neutrophil recruitment
to the peritoneal cavity in the carrageenan induced peritonitis model, what can be confirmed by MPO assay.
Myeloperoxidase (MPO) is an enzyme plentiful existent in neutro-phils granules and is released when these cells are activated, contribut-ing to the pathogenesis of inflammation, and, as its activity is directly linked to neutrophil infiltration in tissues, it is used as an important marker to this event (Santiago et al., 2015).
Our results of the evaluation of MPO activity from peritoneal wash showed that the administration of carrageenan increased the enzyme concentration, while the pre-treatment with LPG (12.5 mg/kg, p.o.) inhibited the increase of MPO activity, reinforcing the proposal that the anti-inflammatory effect of lycopene purified from red guava
in-volves the inhibition of neutrophil infiltration.
Furthermore, MPO catalyzes the oxidation of halide ions, as Cl−, by H2O2,producing acids like, for example, hypochlorous acid, a non-spe-cific oxidant, more toxic than O2−or H2O2(Conner & Grisham, 1996;
Prokopowicz, Marcinkiewicz, Katz, & Chain, 2012; Binder et al., 2013). Thus, the decrease in the enzyme activity also represents an attenuation of oxidative stress.
Additionally, the redox-protector effect of LPG (12.5 mg/kg p.o.) can also be suggested by increased levels of GSH. GSH levels is one of the main markers of oxidative stress related to pathologic processes (Huber, Almeida, & Fátima, 2008; Singh, Karthigesu, Singh, & Kaur, 2014), and can be used to evaluate the redox activity of bioactive mole-cules in the protection against inflammation. Glutathione is a
compo-nent of the cellular antioxidant system. It is a tripeptide that acts in cellular defense against oxidative stress by its sulfhydryl reactive group reducing capability (Çevik et al., 2012; Ahmad, Wani, & Ahsan, 2014).
The antioxidant activity during inflammatory reactions is important because free radicals from leukocytes, principally from neutrophils, cause some types of tissue injuries through the direct degradation of es-sential cellular components, or may start or amplify the inflammatory response through the modulation of the expression of many genes in-volved in the inflammatory process (Conner & Grisham, 1996).
Litera-ture has indicated that molecules with antioxidant activity might influence positively GSH concentrations (Grupta et al., 2011; Parhiz, Roohbakhsh, Soltani, Rezaee, & Iranshahi, 2015). In this study, LPG (12.5 mg/kg, p.o.) significantly increased GSH levels, while carrageenan administration decreased them.
These data indicate that lycopene purified from red guava performs
a redox-protective action during acute inflammation. Indeed, previous studies have showed that lycopene, or fractions rich in lycopene, anti-inflammatory effect is related to its antioxidant properties (Yaping,
Wenli, Weili, & Ying, 2003; Bignotto et al., 2009; Kim et al., 2014; Guo, Liu, & Wang, 2015).
5. Conclusion
In short, considering the results obtained and the experimental con-ditions, it is possible to infer that oral and peritoneal administration of lycopene extract from guava and lycopene purified from guava grants an inhibitory effect on the acute inflammation. The oral administration of lycopene purified from guava has beneficial effect on the inhibition
of leukocyte migration and stabilization of mast cell membrane, as well as on the protection against the effects of oxidative stress, modula-tion of inflammatory mediators and inhibition of genes expression
in-volved in inflammation. These data offer important prospects about the application of lycopene obtained from red guava as an anti-infl am-matory agent.
Acknowledgments
LTDA and Centroflora Group. Adriany Amorim is grateful to CAPES for the doctoral fellowship process n° 99999.004236/2014-09 in Federal University of Piauí - UFPI (RENORBIO program).
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