Anti-inflammatory activity of herb products from Licania rigida Benth_

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<p>Contents lists available at ScienceDirect</p><p>Complementary Therapies in Medicine</p><p>journal homepage: www.elsevier.com/locate/ctim</p><p>Short Communication</p><p>Anti-inflammatory activity of herb products from Licania rigida Benth.</p><p>Enaide Soares Santosa, Cícera Datiane de Morais Oliveiraa, Irwin Rose Alencar Menezesa,</p><p>Emmily Petícia do Nascimentoa, Denise Bezerra Correiaa, Cícero Damon Carvalho de Alencara,</p><p>Maria de Fátima Sousaa, Cícera Norma Fernandes Limaa, Álefe Brito Monteiroc,</p><p>Camila Pedroso Estevam de Souzab, Gyllyandeson de Araújo Delmondesa, Daniel Souza Bezerraa,</p><p>Francisca Adilfa de Oliveira Garciab, Aline Augusti Boligond, José Galberto Martins da Costaa,</p><p>Henrique Douglas Melo Coutinhoa,⁎, Cícero Francisco Bezerra Felipec, Marta Regina Kerntopfa</p><p>a Regional University of Cariri, Brazil</p><p>b The University of western Ontario, Canada</p><p>c Federal University of Paraíba, Brazil</p><p>d Federal University of Santa Maria, Brazil</p><p>A R T I C L E I N F O</p><p>Keywords:</p><p>Licania rigida</p><p>Inflammation</p><p>Flavonoids</p><p>Phenolic acids</p><p>A B S T R A C T</p><p>Purpose: The objective of the present study was to evaluate the systemic anti-inflammatory activity of the hy-</p><p>droalcoholic extract of the leaves of Licania rigida Benth (EHFLR) on models of systemic inflammation in mice.</p><p>Methods: The quantitative chemical profiles of phenolic acids and flavonoids were performed by High-</p><p>Performance Liquid Chromatography (HPLC). Systemic anti-inflammatory activity was determined from car-</p><p>rageenan and dextran-induced paw edema models and the animals were orally treated (p.o.) with EHFLR at</p><p>doses of 25, 50, 100mg/kg, indomethacin (10mg/kg) for carrageenan-induced paw edema and promethazine</p><p>(6mg/kg) for dextran-induced paw edema. The possible mechanisms involved in the anti-inflammatory action of</p><p>the extract were evaluated by the paw edema models induced by histamine and arachidonic acid, and by the</p><p>model of carrageenan-induced peritonitis, where vascular permeability and leukocyte migration to the perito-</p><p>neal cavity were evaluated.</p><p>Results: The results of the HPLC identified the presence of phenolic acids and flavonoids, with chlorogenic acid</p><p>(1.16%) and Caempferol (0.81%) as the main constituents. From the results, it was concluded that the extract</p><p>has an LD50 ≥5000mg/kg when administered orally in mice as this dose did not trigger deaths in any of the</p><p>observed groups. EHFLR (25mg/kg) showed a significant antiderematogenic effect on histamine and arachi-</p><p>donic acid-induced paw edema at the third hour of the tests, with a percentage of inhibition of 46.64% and</p><p>18.33%, respectively. The extract (25mg/kg, p.o.) also significantly reduced vascular permeability and leuko-</p><p>cyte migration in the peritoneal cavity.</p><p>Conclusions: It is concluded that EHFLR exerts a systemic anti-inflammatory action, which seems to depend, at</p><p>least in part, on the inhibition of arachidonic acid metabolism and the action of vasoactive amines. In addition,</p><p>the extract reduced the leukocyte migration in the peritoneal cavity, indicating that its action may be linked to</p><p>the inhibition of pro-inflammatory cytokines.</p><p>1. Background</p><p>Inflammation is a defense response that occurs after cell damage</p><p>caused by microbes, physical agents (radiation, trauma, burns), che-</p><p>micals (toxins, caustic substances), tissue necrosis, or immune reactions</p><p>whose purpose is to promote healing or tissue repair. It is a process</p><p>regulated by pro and anti-inflammatory factors.1 This process triggers a</p><p>series of vascular and cellular events, such as fluid leakage, cell mi-</p><p>gration, and the release of reactive mediators responsible for lysis and</p><p>tissue repair. Depending on the persistence of the lesion and clinical</p><p>signs (flushing, heat, edema, pain and loss of function), this process can</p><p>be characterized as an acute or chronic inflammation.2,3</p><p>https://doi.org/10.1016/j.ctim.2019.06.001</p><p>Received 21 January 2019; Received in revised form 17 May 2019; Accepted 2 June 2019</p><p>⁎ Corresponding author at: Department of Biological Chemistry, Laboratory of Microbiology and Molecular Biology, Regional University of Cariri, Av. Cel. Antônio</p><p>Luiz, 1161. Pimenta, 63105-000 Crato, CE, Brazil.</p><p>E-mail address: hdmcoutinho@gmail.com (H.D. Melo Coutinho).</p><p>Complementary Therapies in Medicine 45 (2019) 254–261</p><p>Available online 04 June 2019</p><p>0965-2299/ © 2019 Elsevier Ltd. All rights reserved.</p><p>T</p><p>There are several drugs used to treat inflammation. These include</p><p>nonsteroidal anti-inflammatory drugs (NSAID’s) and glucocorticoids.</p><p>NSAID’s are inhibitors of the enzyme cyclooxygenase (COX) and can</p><p>produce adverse reactions such as gastritis, gastric ulcers, gastro-</p><p>intestinal perforation, platelet dysfunction, hemorrhage and renal im-</p><p>pairment.4 On the other hand, glucocorticoids cause some undesirable</p><p>symptoms, such as irritability and insomnia, which occur with short</p><p>administrations. Other more serious effects may be Cushing's syndrome,</p><p>osteoporosis, osteonecrosis, growth retardation, dyslipidemia, hy-</p><p>pertension, thrombosis, vasculitis, behavioral changes, as well as in-</p><p>creased risk of serious infections triggered with continued use of this</p><p>drug.5</p><p>The search for new drugs has stimulated researchers and the phar-</p><p>maceutical industry to invest more in the research of medicinal plants</p><p>in order to investigate the substances involved in their therapeutic ac-</p><p>tion. Thus, this plants may be used as raw material for the sythesis of</p><p>pharmacologically active compounds, thereby highlighting its im-</p><p>portance in the search of new drugs development.6</p><p>The species L. rigida of the Chrysobalanaceae family, commonly</p><p>known as "oiticica", can be found in the Brazilian states of Piauí, Rio</p><p>Grande do Norte, Ceará (typical of riparian forests of the Sertão</p><p>Northeast) and Paraíba. Various parts of the EHFLR are used in folk</p><p>medicine. Its leaves are popularly used in the treatment of in-</p><p>flammatory processes,7 including inflammation of the male and female</p><p>reproductive system.8 In addition, leaves are also employed in the</p><p>treatment of diabetes.9 It has also been reported that the seeds present</p><p>antioxidant and antibacterial action for Staphylococcus aureus, besides</p><p>having an antidiarrheal and healing action.10 The dry bark also has</p><p>antidiarrheal and antispasmodic action. This last activity is possibly due</p><p>to the presence of tannins, flavonoids and saponins and quinones in</p><p>their composition.8</p><p>Although L. rigida is widely used by the population of Northeastern</p><p>Brazil for medicinal purposes, there is still a lack of studies to in-</p><p>vestigate the pharmacological properties of the genus Licania, which</p><p>can serve as a source for molecules with a potential anti-inflammatory</p><p>effect.11 Thus, the objective of the present study was to evaluate the</p><p>systemic anti-inflammatory activity of the hydroalcoholic extract of the</p><p>leaves of L. rigida (oiticica) in mice.</p><p>2. Methods</p><p>2.1. Collection and preparation of the hydroalcoholic extract from the</p><p>leaves of Licania rigida Benth</p><p>The leaves of L. rigida were collected in Missão Velha - CE, Brazil (7°</p><p>17′29.8″S, 39° 10′ 42.4″W, altitude: 377m) in February 2016 under the</p><p>authorization of the Chico Mendes Institute for Conservation of</p><p>Biodiversity - ICMBio (nº 54896-1). The species was identified by Dr.</p><p>Maria Arlene Pessoa da Silva in the Caririense Dárdano de Andrade</p><p>Lima Herbarium (HCDAL) of the Regional University of Cariri (URCA)</p><p>under registration number 12,544.</p><p>After collection, the leaves (1.452 g) were washed, exposed to shade</p><p>drying, macerated and submitted to hydroalcoholic extraction (1:1) for</p><p>72 h. Then the extractive solution was filtered and subjected to dis-</p><p>tillation in a rotary evaporator apparatus for the removal of the ethanol.</p><p>The resulting solution was frozen (−20 °C) and subjected to lyophili-</p><p>zation, obtaining a yield of 27.14%.</p><p>2.2. Chemical tests</p><p>2.2.1. Chemical prospecting</p><p>The extract was submitted to qualitative chemical tests according to</p><p>the methodology of Matos. 12 The tests were based on the visual ob-</p><p>servation of the colorimetric change and/or accumulation of precipitate</p><p>after the addition</p><p>of specific reagents for each chemical class present in</p><p>the extract.</p><p>2.2.2. Quantification of phenolic acids and flavonoids by High Performance</p><p>Liquid Chromatography (HPLC-DAD)</p><p>The chromatographic analysis of the EHFLR was performed on a</p><p>Shimadzu Prominence LC-20AT apparatus, with alternating pumps to</p><p>the DGU 20A5 degasser, SPD-M20A Diode Array Detector (DAD) in-</p><p>tegrator and SP1 LC 1.22 software solution.</p><p>Reverse phase chromatographic analyzes were performed under</p><p>gradient conditions using C18 column (4.6mm x 250mm) packed with</p><p>particles of 5 μm in diameter; for the mobile phase it was used water</p><p>containing 2% acetic acid (A) and acetonitrile (B). The composition</p><p>gradient was: 5% (B) for 2min; 25% (B) in 10min; 40, 50, 60, 70 and</p><p>80% (B) every 10min; following the method described by Bitencourt</p><p>et al 14 with minor modifications. The L. rigida extract and the mobile</p><p>phase were filtered through a 0.45 μm membrane (Millipore), degassed</p><p>by ultrasonic bath, and the extract was analyzed at a concentration of</p><p>15mg/mL. The flow rate was 0.6mL/min. and the injection volume</p><p>was 40 μL. Standard solutions were prepared in the mobile phase of</p><p>HPLC in the concentration range of 0.030 to 0.500mg/mL. Quantifi-</p><p>cation was performed by integrating the peaks using the standard</p><p>method at 254 nm for gallic acid, 280 nm for catechin and epicatechin,</p><p>326 nm for chlorogenic and caffeic acids and 365 for quercetin, rutin,</p><p>caempferol. Chromatographic peaks were confirmed comparing with</p><p>the retention time of the reference standards and the DAD spectra</p><p>(200–600 nm). All chromatographic analyzes were performed at room</p><p>temperature and in triplicate.</p><p>2.3. In vivo experimental protocols</p><p>2.3.1. Drugs</p><p>Evans blue, carrageenan, dexamethasone, dextran, histamine and</p><p>promethazine were purchased from Sigma-Aldrich (St. Louis, MO,</p><p>USA). Arachidonic acid and indomethacin were purchased from</p><p>Dynamics (Brazil) Merck Sharp & Dohme (Brazil), respectively. All so-</p><p>lutions were prepared immediately prior to use. EHFLR was diluted in</p><p>saline and administered orally (p.o.).</p><p>2.3.2. Animals and ethical aspects of the study</p><p>Swiss albino mice (Mus musculus), male, weighing between 25–30 g</p><p>were used. The animals were kept in polypropylene cages in controlled</p><p>environment with a light/dark 12 h cycle, temperature of 22 ± 2 °C,</p><p>humidity of approximately 70%, with free access to drinking water and</p><p>feed (Labina, Purina). The animals were fasted (3–4 hrs) before each</p><p>test. All the experimental procedures followed the norms of animal use,</p><p>with the research being submitted and approved by the Committee of</p><p>Ethics in Animal Research of the Regional University of Cariri (CEUA/</p><p>URCA), with protocol number 30/2016.2.</p><p>2.3.3. Estimated median lethal dose (LD50)</p><p>The oral LD50 of the EHFLR followed an adaptation of OECD 425</p><p>(OECD, 2008).13 The mice were randomly divided into groups com-</p><p>posed of four animals; the control group received saline (0.9%) and the</p><p>others received EHFLR at doses of 1.75, 5.5, 17.5, 55, 175, 550, 2000</p><p>and 5000mg/kg. Then the animals were observed for 10, 20, 30, 60,</p><p>120, 180 and 240min and for 14 consecutive days after treatment,</p><p>observing the occurrence of signs of toxicity14 and lethality.</p><p>2.3.4. Anti-inflammatory activity</p><p>The acute anti-inflammatory activity of the extract was evaluated</p><p>from paw edema models (induced by different phlogistic agents,</p><p>namely carrageenan, dextran, histamine and arachidonic acid), and</p><p>carrageenan-induced peritonitis, for which vascular permeability and</p><p>the migration of leukocytes into the peritoneal cavity were evaluated.</p><p>The animals were divided into three groups composed of six mice each:</p><p>negative control (saline 0.9%, p.o.), positive control (indomethacin</p><p>10mg/kg, p.o., dexamethasone 4mg/mL/p.o., promethazine 6mg/kg),</p><p>and EHFLR (25, 50, 100mg/kg, p.o.). In the paw edema tests, each</p><p>E.S. Santos, et al. Complementary Therapies in Medicine 45 (2019) 254–261</p><p>255</p><p>animal had the initial volume (Vi) of the right/left hind paw evaluated</p><p>before the start of treatment. The expression of the results was obtained</p><p>through the difference between the final volume (Vf) and the initial</p><p>volume (Vi) of the legs at each moment, calculated in the formula:</p><p>Ve=Vf - Vi, where Ve= edema volume.</p><p>2.3.5. Paw edema induced by intra-plantar injection of carrageenan 1%</p><p>Mice were pretreated with saline (0.9%, p.o.), indomethacin</p><p>(10mg/kg, p.o.) and EHFLR (25, 50 and 100m/kg, p.o.). After 1 h,</p><p>each animal received an intraplantar injection of 1% carrageenan</p><p>(20 μL/paw) in the right hind paw and saline in the left paw. The hind</p><p>paw volume was recorded after 1, 2, 3 and 4 h of the phlogistic agent</p><p>injection.15</p><p>2.3.6. Paw edema induced by intra-plantar injection of dextran 1%</p><p>Mice were pretreated with saline (0.9%, p.o.), promethazine (6mg/</p><p>kg, p.o.) and EHFLR (25, 50 and 100mg/kg, p.o.). After 1 h, each an-</p><p>imal received an intraplantar injection of 1% dextran (20 μL/paw) in</p><p>the right hind paw and saline in the left paw. The volume of the right</p><p>and left hind paw of each animal was recorded after 1, 2, 3 and 4 h of</p><p>injection of the phlogistic agent dextran.15</p><p>2.3.7. Paw edema induced by intra-plantar injection of histamine 1%</p><p>Mice were pretreated with saline (0.9%, p.o.), promethazine (6mg/</p><p>kg, p.o.) and EHFLR (25mg/kg, p.o.). After 1 h, each animal received</p><p>an intraplantar injection of 1% histamine (20 μL) in the right hind paw</p><p>and saline in the left paw. The volume of the hind paws of each animal</p><p>was recorded 30min, 60min, 90min and 120min after injection of the</p><p>phlogistic agent.16</p><p>2.3.8. Paw edema induced by intra-plantar injection of arachidonic acid</p><p>1%</p><p>Mice were pretreated with saline (0.9%, p.o.), promethazine (6mg/</p><p>kg, p.o.) and EHFLR (25mg/kg, p.o.). After 1 h each animal received an</p><p>intraplantar injection of 1% arachidonic acid (20 μL) in the right hind</p><p>paw and saline in the left paw. The volume of the hind paws of each</p><p>animal was recorded 15min, 30min, 45min and 60min after the in-</p><p>jection of the phlogistic agent.16</p><p>2.3.9. Vascular permeability analysis by Evans blue extravasation</p><p>Mice were treated with saline (0.9%, p.o.), dexamethasone (5mg/</p><p>kg, p.o.) and EHFLR (25mg/kg, p.o.). After 60min of treatment, each</p><p>rodent received an intraperitoneal injection of 1% carrageenin and</p><p>200 μL of Evans blue (0.2 mL) through the retro orbital plexus. After</p><p>4 h, autase was performed, which was followed by the administration of</p><p>a 3mL volume of PBS in the peritoneal cavity of the animals. A gentle</p><p>massage was then performed on the abdominal region of the mice and</p><p>for collection of the peritoneal fluid (1.5–2mL). Afterwards, the sam-</p><p>ples were centrifuged for 2min at 6000 rpm, for subsequent reading of</p><p>the supernatant in a spectrophotometer, with a wavelength of</p><p>520 nm.15</p><p>2.3.10. Carrageenan-induced peritonitis</p><p>Mice were treated with saline (0.9%, p.o.), dexamethasone (5mg/</p><p>kg, p.o.) and EHFLR (25mg/kg, p.o.). After 60min of treatment, each</p><p>rodent received 1% carrageenan intraperitoneal injection. After 4 h, the</p><p>autase was performed, followed by the administration of a 3mL volume</p><p>of heparinized PBS in the peritoneal cavity of the animals. Then a gentle</p><p>massage was performed on the abdominal region of the mice and the</p><p>peritoneal fluid was collected for a subsequent leukocyte count, which</p><p>was performed by the ABX Micros 60 device.15</p><p>2.3.11. Statistical analysis</p><p>The results were expressed as mean ± standard error of the mean</p><p>(SEM). For comparison of procedures, one-way or two-way ANOVA was</p><p>applied followed by post hoc Bonferroni test. All data were analyzed by</p><p>the software GraphPad Prism (version 5.0). The differences observed</p><p>were considered significant when p<0.05.</p><p>3. Results</p><p>3.1. Chemical prospecting</p><p>The qualitative chemical profile was observed by the presence of</p><p>classes of secondary metabolites present in the EHR according shown in</p><p>Table 1.</p><p>3.2. Quantification of phenolic acids and flavonoids by High Performance</p><p>Liquid Chromatography (HPLC / DAD)</p><p>The quantitative analysis of phenolic acids and flavonoids present</p><p>in</p><p>the extract was performed by the HPLC method. The results of the the</p><p>quantification of the following phenolic acids: gallic acid (0.42%),</p><p>chlorogenic acid (1.16%) and caffeic acid (0.43%), and the following</p><p>flavonoids: epicatechin (0.20%), rutin (0.08%), quercetin (0.43%),</p><p>caempferol (0.81%) and catechin (0.01%) as shown in Table 2 and</p><p>Fig. 1. This result was corroboring with qualtitative analysis that shows</p><p>the presence of the Catechins, Chalconas, flavonoids and tannins.</p><p>3.3. Median lethal dose (LD50)</p><p>Oral administration of the hydroalcoholic extract from the leaves of</p><p>Licania rigida (EHFLR) showed no signs of toxicity or lethality, and its</p><p>estimated LD50 was higher than 5000mg/kg, p.o. For the pharmaco-</p><p>logical tests, doses were determined respecting the limit of 10% of LD50.</p><p>Therefore, the doses used were 25, 50 and 100mg/kg of the EHFLR.</p><p>Table 1</p><p>Chemical prospection of the hydroalcoholic extract from</p><p>Licania rigida Benth’s leaves.</p><p>Secondary metabolites groups EHFLR</p><p>Anthocyanidins –</p><p>Anthocyanins –</p><p>Auronas +</p><p>Catechins +</p><p>Chalconas +</p><p>Flavones +</p><p>Flavonols +</p><p>Flavonones +</p><p>Leucoantocyanidin +</p><p>Tannins catechic +</p><p>Xanthones +</p><p>Alkaloids –</p><p>EHFLR: Hydroalcoholic extract of the leaves of Licania rígida</p><p>Benth, +: Present, –: Absent.</p><p>Table 2</p><p>Quantification of the phenolic and flavonoid compounds in the hydroalcoholic</p><p>extract from Licania rigida Benth’s leaves.</p><p>Compounds EHFLR</p><p>Concentration (mg/g) Percentage (%)</p><p>Gallic acid 4.29 ± 0.01 0.42</p><p>Catechin 0.18 ± 0.01 0.01</p><p>Chlorogenic Acid 11.65 ± 0.03 1.16</p><p>Caffeic acid 4.37 ± 0.04 0.43</p><p>Epicatechin 2.03 ± 0.02 0.20</p><p>Rutin 0.86 ± 0.05 0.08</p><p>Quercetin 4.39 ± 0.02 0.43</p><p>Kaempferol 8.15 ± 0.01 0.81</p><p>EHFLR: Hydroalcoholic extract of the leaves of Licania rígida Benth.</p><p>E.S. Santos, et al. Complementary Therapies in Medicine 45 (2019) 254–261</p><p>256</p><p>3.4. Paw edema induced by intra-plantar injection of carrageenan 1%</p><p>The results show that the extract was able to reduce edema induced</p><p>by 1% carrageenin in the third hour of the test at all doses analyzed,</p><p>showing statistical significance with a percentage of reduction of</p><p>29.09% at the dose of 25mg/kg, 29.4%; at a dose of 50mg/kg, and</p><p>22.26% at the dose of 100mg/kg, in relation to the control group. As</p><p>expected, indomethacin was effective in reducing the volume of edema</p><p>significantly at all time points analyzed in relation to the saline group</p><p>(1 h: 44.1%, 2 h: 34.82%, 3 h: 34.89%, 4 h: 32.17% are the percentages</p><p>of reduction for times), with a higher percentage of inhibition at the 3rd</p><p>hour of the test (Fig. 2). Then, the results no showed the dose-depen-</p><p>dence effect.</p><p>3.5. Paw edema induced by intra-plantar injection of dextran 1%</p><p>The results showed that EHFLR reduced the volume of edema in-</p><p>duced by dextran significantly in relation to the saline group at the dose</p><p>of 25mg/kg in in all times analyzed (1 h: 62.3%, 2 h: 52.4%, 3 h: 44,</p><p>5%, 4 h: 41.2%), and was statistically significant from the 1 st hour. On</p><p>the other hand, administration of the extract at the dose of 100mg/kg</p><p>and 50mg/kg did show lower efficacy in reduction of the edema vo-</p><p>lume. Promethazine (6mg/kg) used as the standard drug was able to</p><p>significantly inhibit edema from the 1 st hour of evaluation until the 4th</p><p>hour (1 h: 78.1%, 2 h: 70.4%, 3 h: 47.7%, 4 h: 49.17%) when compared</p><p>to the saline solution only group (Fig. 3).</p><p>3.6. Paw edema induced by intra-plantar injection of histamine 1%</p><p>The dose of 25mg/kg present better efficacy in screening assay by</p><p>carrageenan and dextran, then, the dose was used to evaluation of</p><p>pathway involved in the anti-inflammatory effect. According to Fig. 4,</p><p>EHFLR at a dose of 25mg/kg significantly reduced paw edema in the</p><p>animal significantly at all times analyzed (1 h: 19.71%, 2 h: 39.14%,</p><p>3 h: 46.64%, 4 h: 5.34%), showing a higher percentage of inhibition at</p><p>the 3rd hour. Similarly, promethazine (6mg/kg), an antihistamine</p><p>drug, significantly reduced edema over the saline group at all time</p><p>points (1 h: 36.29%, 2 h: 46.26%, 3 h: 49.26%, 4 h: 18.42%). This result</p><p>was corrobate with dextran assay.</p><p>3.7. Paw edema induced by intra-plantar injection of arachidonic acid 1%</p><p>The results showed that the dose of 25mg/kg EHFLR showed a</p><p>significant reduction in the volume of edema in relation to the saline</p><p>group only at times 1 h (16.83%), 2 h (12.49%) and 3 h (18.33%).</p><p>Indomethacin (10mg/kg), standard anti-inflammatory drug, reduced</p><p>the volume of edema from the first minutes of evaluation (1 h: 22.85%,</p><p>2 h: 52.22%, 3 h: 44.71%, 4 h: 32.17%), compared to the salt group</p><p>(Fig. 5).</p><p>Fig. 1. Phenolic compounds and flavonoids presents in the hydroalcoholic ex-</p><p>tract from Licania rigida Benth’s leaves.</p><p>Gallic acid (peak 1), Catechin (peak 2), Chlorogenic acid (peak 3), caffeic acid</p><p>(peak 4), epicatechin (peak 5), rutin (peak 6), quercetin (peak 7) and caemp-</p><p>ferol (peak 8).</p><p>Fig. 2. Anti-inflammatory effect of the hydroalcoholic extract from Licania ri-</p><p>gida Benth’s leaves in the carrageenan-induced paw edema test.</p><p>The columns represent the mean ± standard error of the mean (SEM) related</p><p>to the % of edema. Differences in the mean were assessed two-way ANOVA</p><p>followed by Bonferroni (n= 6 mice per group). At each time point, significant</p><p>diferences between treatment and control group (saline) are highlighted using</p><p>the letter a, where a1 means p < 0.05; a2 p < 0.01; a3 p < 0.01; a4</p><p>p < 0.01. EHFLR: hydroalcoholic extract of the leaves of Licania rigida Benth.</p><p>Fig. 3. Anti-inflammatory effect of the hydroalcoholic extract from Licania ri-</p><p>gida Benth’s leaves in the dextran-induced paw edema test.</p><p>The columns represent the mean ± standard error of the mean (SEM), ana-</p><p>lyzed by two-way ANOVA followed by Bonferroni (n=6 mice per group). The</p><p>values were considered significant when a4 = p < 0.01 vs saline. EHFLR:</p><p>hydroalcoholic extract of the leaves of Licania rigida Benth.</p><p>Fig. 4. Anti-inflammatory effect of the hydroalcoholic extract from Licania ri-</p><p>gida Benth’s leaves in the histamine-induced paw edema test.</p><p>The columns represent the mean ± standard error of the mean (SEM), ana-</p><p>lyzed through the two-way ANOVA followed by Bonferroni (n= 6 mice per</p><p>group). Significant values of a3 = p < 0.01; a4 = p < 0.01 vs saline. EHFLR:</p><p>hydroalcoholic extract of the leaves of Licania rigida Benth, ns: no significant.</p><p>E.S. Santos, et al. Complementary Therapies in Medicine 45 (2019) 254–261</p><p>257</p><p>3.8. Vascular permeability by Evans blue extravasation</p><p>The results show that EHFLR inhibited the increase of vascular</p><p>permeability induced by carrageenan in a percentage of 57.14%, re-</p><p>lative to the saline group. Dexamethasone significantly reduced vas-</p><p>cular permeability by 61.90% in relation to the saline group (Fig. 6).</p><p>3.9. Carrageenan-induced peritonitis</p><p>In this test, the extract reduced neutrophil migration significantly</p><p>compared to the saline treated group in 47.8%. Likewise, the extract</p><p>reduced the migration of monocytes significantly in relation to the</p><p>saline treated group in 43.83%. Dexamethasone significantly reduced</p><p>the migration of neutrophils and monocytes in 59.29% and 68.94%,</p><p>respectively, in relation to the saline treated group (Fig. 7A and 7B).</p><p>4. Discussion</p><p>The chemical classes identified in the chemical analysis of the</p><p>EHFLR resemble, in part, the results obtained by Lonrenze17 who, when</p><p>evaluating the chemical profile of the same species, identified the</p><p>presence of fatty acids, tannins, terpenes and flavonoids. Braca et al.18</p><p>also demonstrate that chemical studies of the species of this genus</p><p>present flavonoids in their composition, corroborating with the present</p><p>study that shares the presence of several types of flavonoids.</p><p>The study by Castilho, Oliveira and Kaplan19 share some of the same</p><p>findings when revealing that species of the genus Licania present as</p><p>main classes of secondary metabolites flavonoids and triterpenes (not</p><p>identified in the present study). The flavonoids are widely distributed</p><p>Fig. 5. Anti-inflammatory effect of the hydroalcoholic extract from Licania ri-</p><p>gida Benth’s leaves in the paw edema test induced</p><p>by arachidonic acid.</p><p>The columns represent the mean ± standard error of the mean (SEM), ana-</p><p>lyzed by two-way ANOVA followed by Bonferroni (n= 6 mice per group). The</p><p>values were considered significant when a4 = p < 0.01 vs saline. EHFLR:</p><p>hydroalcoholic extract of the leaves of Licania rigida Benth.</p><p>Fig. 6. Effect of the hydroalcoholic extract from Licania rigida Benth’s leaves on</p><p>the peritoneal-extravasation of Evans blue test in mice (For interpretation of the</p><p>references to colour in this figure legend, the reader is referred to the web</p><p>version of this article).</p><p>The columns represent the mean ± standard error of the mean (SEM), ana-</p><p>lyzed by one-way ANOVA followed by Bonferroni (n= 6 mice per group). The</p><p>values were considered significant when a4 = p < 0.01 vs Saline. EHFLR:</p><p>hydroalcoholic extract of the leaves of Licania rigida Benth.</p><p>Fig. 7. Effect of the hydroalcoholic extract from Licania rigida Benth’s Leaves on</p><p>the neutrophils and monocytes migration in the carrageenan-induced perito-</p><p>nitis test.</p><p>The columns represent the mean ± standard error of the mean (SEM), ana-</p><p>lyzed by one-way ANOVA followed by Bonferroni (n=6 mice per group). The</p><p>values were considered significant when a4 = p < 0.01 vs saline. (A)</p><p>Percentage of Granulocytes and (B) percentage of Monocytes. EHFLR: hydro-</p><p>alcoholic extract of the leaves of Licania rigida Benth.</p><p>E.S. Santos, et al. Complementary Therapies in Medicine 45 (2019) 254–261</p><p>258</p><p>throughout the plant kingdom, possessing many biological actions,</p><p>among them antitumor, antiulcer, antioxidant, antiviral and anti-in-</p><p>flammatory action.20</p><p>The HPLC identified the presence of the following secondary me-</p><p>tabolites: gallic acid, chlorogenic acid, caffeic acid, epicatechin, rutin,</p><p>quercetin, caempferol and catechin. Teixeira et al10 have analysed the</p><p>hydroalcoholic extract from the shell and pulp of Licania tomentosa</p><p>Benth Fristch species, which shares the same genus of the plant under</p><p>study, where it showed compounds similar to those identified in our</p><p>study. The shell has presented compounds such as gallic acid, catechin,</p><p>rutin and kaempferol, on the other hand the pulp has presented gallic</p><p>acid and kaempferol. Braca et al.18 performed this analysis with dif-</p><p>ferent species of the genus Licania - Licania apetalada (E.Mey.), Licania</p><p>densiflora Hook.f., Licania heteromorfa Var., Licania pittieri Prance, Li-</p><p>cania pyrifolia Griseb and Licania carii Cardozo), and showed that they</p><p>had the presence of some flavonoid derivatives such as caempferol,</p><p>quercetin and myricetin.</p><p>Scientific studies that prove the toxicity of previously unrestricted</p><p>and indiscriminately used plants are in increasing numbers.22 As there</p><p>are no specific reports on the toxicity of the hydroalcoholic extract of</p><p>the leaves of L. rigida, it was necessary to evaluate its possible acute</p><p>toxicity by estimating the LD50 concomitantly with the Hippocratic</p><p>screening before starting the experimental trials.</p><p>Studies by Teixeira 11 have demonstrated that the leaf extract of L.</p><p>rigida and Licania tomentosa Benth do not induce biochemical changes</p><p>or oral toxicity in the LD50 test. This study did not reveal any death</p><p>during the 14 days of evaluation, with LD50 above 2000mg/kg. This</p><p>fact corroborates with the present study that evaluated the lethal dose</p><p>of EHFLR and obtained similar results as it did not evidence deaths</p><p>during the same follow-up period. With the Hippocratic screening it</p><p>was also possible to perceive that the extract was devoid of any signs of</p><p>toxicity.</p><p>Intra-plantar injection of 1% carrageenan induced an acute and</p><p>progressive increase in injected paw volume. This edema is propor-</p><p>tional to the intensity of the inflammatory response constituting a</p><p>useful parameter in the evaluation of anti-inflammatory drug activity.23</p><p>The inflammatory edema induced by carrageenan is the result of se-</p><p>quential action and integrates several inflammatory mediators.24</p><p>Following administration of carrageenan there is extravasation of</p><p>plasma proteins and recruitment of neutrophils to the inflamed site.</p><p>Initially, the release of vasoactive amines characterizing the initial</p><p>phase of inflammation is observed; Later, in the late phase, cytokines</p><p>and prostaglandins, especially PGE2, are released. This process is in-</p><p>tensified during the 3rd hour of the test, characterized as the peak of</p><p>inflammation. After this time, the inflammation reduces its response</p><p>and consequently edema,25 which justifies the choice in emphasizing</p><p>the statistical values of the present work to the peak of the 3rd hour.</p><p>Studies performed by Dos Santos et al 26 show that chlorogenic acid,</p><p>present in species of the studied family, has inhibited carrageenan-in-</p><p>duced paw edema in rats, corroborating with the findings presented in</p><p>this study. Studies that investigated the anti-inflammatory effect of</p><p>chlorogenic acid, stated that its possible mechanisms mediating in-</p><p>flammation reduction occur due to the inhibition of: TLR4 signaling</p><p>pathway in carbon tetrachloride (CCl4)-induced liver fibrosis27; LPS-</p><p>inflamed keratinocytes28; decreased nitric oxide (NO) production</p><p>mediated by down-regulation of iNOS; up-regulation of pro-in-</p><p>flammatory cytokines such as IL-1β, TNF-α, and IL-6, as well as the</p><p>chemokine CXCL1 through down-regulation of NF-κB and inhibition of</p><p>Ninj1, which is important for leukocyte infiltration.29</p><p>Studies conduced by Hämäläinen et al 30 using human hepatocyte-</p><p>derived cell line Chang Liver demonstrate that kaempferol can mod-</p><p>ulate inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)</p><p>and reactive C-protein (CRP) expression, and to induce changes in the</p><p>nuclear factor kappa B (NF-κB) pathway.</p><p>García-Mediavilla et al 31 using Murine J774 macrophages from</p><p>American Type Culture Collection demonstrate that kaempferol and</p><p>quercetin inhibited activation of important transcription factors for</p><p>iNOS, that is, STAT-1/STAT-3 and NF-κB and can partly explain the</p><p>anti-inflammatory effect envolded of these flavonoids. The kaempferol</p><p>deactivated and prevented formaton of two major transcription factors</p><p>STAT3 and NF-κB and, attenuated IL-6-induced COX-2 expression in</p><p>acute inflammation by carrageenan-induced mouse paw.32</p><p>Guardia et al 33, when investigating the anti-inflammatory proper-</p><p>ties of flavonoids, observed that rutin and quercetin (compounds</p><p>identified in EHFLR) have shown to be effective in reducing carra-</p><p>geenan-induced paw edema. Fawzy, Vishwanath and Franson 34 ob-</p><p>served that quercetin inhibits, in a dose-dependent manner, the release</p><p>of [14C] oleate from labeled phospholipid coused only by the extra-</p><p>cellular phospholipase A2 (PLA2).</p><p>Dextran is a polysaccharide used in inflammation tests to promote</p><p>the release of histamine and serotonin from mast cells during edema</p><p>formation. These substances alter the tone and vascular permeability,</p><p>contributing to fluid extravasation.35 Similar to carrageenan-induced</p><p>paw edema, the induction of edema with dextran causes an increase in</p><p>vascular permeability, but when the phlogistonic agent is dextran, this</p><p>increase occurs due to degranulation of the mast cells and release of the</p><p>vasoactive amines.</p><p>The vasoactive amines are available in preformed reserves to be</p><p>released during the inflammatory process. Histamine is considered the</p><p>main mediator in the initial phase after injury, increasing vascular</p><p>permeability and acting on the microcirculation. 36 Serotonin, another</p><p>substance involved in this process, induces edema and extravasation of</p><p>plasma during acute inflammation, being released mainly by mast cells</p><p>and platelets. 37 In addition, dextran promotes rapid extravasation of</p><p>fluid with few proteins and cells, unlike the induction of carrageenan</p><p>edema.38</p><p>Promethazine, an antihistamine drug, acts on the inflammatory</p><p>process possibly by decreasing the expression of vasoactive amines and</p><p>thereby contributing to the decrease of edema.37,38 EHFLR, similarly,</p><p>may be acting to reduce cytokine expression and release of histamine</p><p>and serotonin, as well as the standard promethazine drug.39 The</p><p>study</p><p>by Gaind and Gupta 40 shows that flavonoids play important anti-in-</p><p>flammatory activity on the dextran-induced paw edema model in rats at</p><p>the dose of 0.5 g/kg. Flavonoid compounds are reported to affect</p><p>nonspecific immune responses (ivolved in the inflammatory response),</p><p>suppressing macrophagic phagocytosis, mast cell activation, and the</p><p>oxidants release by neutrophils.33</p><p>The species Aniba riparia (Ness.) has flavonoids in its chemical</p><p>composition and it was tested the anti-inflammatory effect of its major</p><p>component in a model of histamine-induced paw edema, resulting in an</p><p>antiedematogenic effect of this species.41 This study corroborates our</p><p>findings because although they are from species of distinct families,</p><p>they present flavonoids in their composition, and play anti-in-</p><p>flammatory activity in similar experimental models.</p><p>Arachidonic acid (AA) is a fatty acid that plays an important role in</p><p>cellular physiology. It is released when there is damage from membrane</p><p>phospholipids through the enzyme phospholipase A2, which can be</p><p>activated by various chemical, inflammatory or traumatic stimuli, in</p><p>addition to activating pro-inflammatory cytokines.42 In response to</p><p>some stimulation, phospholipase hydrolyzes the membrane phospholi-</p><p>pids, releasing arachidonic acid. Arachidonic acid will give rise to in-</p><p>flammatory mediators via the cyclooxygenase (COX) pathway, produ-</p><p>cing prostaglandins (PG’s), prostacyclins (PIs) and thromboxanes (TXs),</p><p>and via lipoxygenases (LOX), leading to leukotriene (LT’s) formation.</p><p>All these molecules are called prostanoids and play a fundamental role</p><p>in the inflammatory process.42</p><p>Non-steroidal anti-inflammatory drugs, such as indomethacin, in-</p><p>hibit cyclooxygenase, reducing prostaglandin biosynthesis, thereby re-</p><p>ducing the inflammatory process.43 In our study, the extract tested</p><p>showed similar action to that of indomethacin in the model of arachi-</p><p>donic acid-induced paw edema. The results of this model corroborate</p><p>the effects of EHFLR on carrageenan-induced paw edema.</p><p>E.S. Santos, et al. Complementary Therapies in Medicine 45 (2019) 254–261</p><p>259</p><p>The methodology used in the analysis of capillary permeability was</p><p>done by measuring the extravasation of the Evans blue dye from the</p><p>vessels into the peritoneal cavity.44 This model allows quantitative</p><p>evaluation of vascular permeability during an inflammatory process.</p><p>This evaluation is done through the intravenous administration of</p><p>Evans blue dye, which has a strong affinity for albumin, which allows</p><p>the evaluation of vascular permeability. If the endothelium is injured,</p><p>consequently the concentration of albumin in the peritoneal lavage will</p><p>be higher.45 Furthermore, this methodology induce acute inflamma-</p><p>tion, which stimulate the release of mediators such as histamine and</p><p>serotonin, important in vasodilation and increased vascular perme-</p><p>ability. Additionally, there is also a prostaglandin E2 release, an im-</p><p>portant smooth muscle vasodilator, responsible for increase the blood</p><p>flow to the site of inflammation and consequently the vascular per-</p><p>meability, resulting in fluid extravasation.46</p><p>The results suggest that the secondary metabolites identified in the</p><p>EHFLR’s chemical test act inhibiting vascular permeability, corrobor-</p><p>ating with the study carried out by Ferreira, 47 which demostrated the</p><p>Cleome spinosa anti-inflammatory activity, induced by the methanolic</p><p>extract from its aerial parts rich in flavonoids, in the pleurisy test in-</p><p>duced by carrageenan in mice. This extract, was capable of inhibiting</p><p>protein extravasation and reducing leukocyte migration to the pleural</p><p>cavity, commonly observed in the pleurisy test. Thus, although Cleome</p><p>spinose and EHFLR are different species, they have similar chemical</p><p>compounds, which support, in part, the results demonstrated in the</p><p>present study.</p><p>TNFa and IL-1b has been pivotal inflammatory cytokine envolved in</p><p>inflammatory response. The TNF-a affects vascular endothelial cells</p><p>that promoting disruption of cell-cell junctions increased vascular</p><p>permeability. Chlorogenic acid and caffeic acid was showed a dose-</p><p>dependent anti-inflammatory effect by decrease suppression of pro-in-</p><p>flammatory cytokine tumor necrosis factor-alpha (TNF-α), expression</p><p>of NF-κB and suppression of ERK1/2, STAT3 and JNK1/2. 48</p><p>Kaempferol significantly reduced the overproduction of TNF‑α,</p><p>IL‑1β, interleukin‑6, ICAM‑1 and VCAM‑1 induced by LPS, indicating</p><p>the negative regulation of kaempferol in TLR4, NF‑κB and STAT sig-</p><p>naling underlying intestinal inflammation.49 Then, the anti-inflamatory</p><p>and reductions of vascular permeability, in part, can explain by che-</p><p>mical profile present in EHFLR.</p><p>Carrageenan-induced peritonitis is a model that involves cell mi-</p><p>gration and plasma exudation into the peritoneal cavity during an in-</p><p>flammatory process. 50 Neutrophils (polymorphonuclear), as well as</p><p>macrophages, are the major phagocytic cells of the immune system.</p><p>During the inflammatory process, the neutrophils are recruited to the</p><p>inflammatory focus playing a defensive role to the organism. 51 This</p><p>model evaluates an acute inflammation, allowing quantification of</p><p>leukocytes that migrate to the peritoneal cavity. This migration occurs</p><p>under the action of chemotactic agents, mainly leukotrienes and in-</p><p>terleukins. The process of migration and passage of leukocytes to the</p><p>inflamed site is dependent on the expression of adhesion molecules (E,</p><p>P, and L-selectin). These molecules regulate leukocyte adhesion medi-</p><p>ated by immunoglobulins and β-2 integrins. This event is determined by</p><p>the action of chemotactic mediators as cytokines (TNF-alpha, inter-</p><p>leukin (IL)-1β and IL-6), nitric oxide, peptide fragments of the com-</p><p>plement system (C3a, C4a and C5a), histamine, leukotrienes, pros-</p><p>taglandins, thromboxane and PAF.52 As previously shown, Chlorogenic</p><p>acid and caffeic acid, rutin and kaempferol can be interfere in expres-</p><p>sions of these mediators. This suggests that the action of EHFLR in re-</p><p>ducing cell migration may be associated with inhibition of the bio-</p><p>synthesis of the pro-inflammatory mediators observed in acute</p><p>inflammation. 53</p><p>Carvalho et al 54 described that the biological activity of flavonoids</p><p>is mainly related to the inhibition of inflammatory mediators. This</p><p>justifies, at least in part, the results of the present study which shows in</p><p>several models of inflammation that EHFLR possibly acts in a systemic</p><p>way: decreasing the expression of vasoactive amines, inhibiting the</p><p>cyclooxygenase enzyme, in addition to reducing the leukocyte migra-</p><p>tion, the major cells to migrate to the peritoneum in the early stage of</p><p>inflammation, according to Fröde-Saleh, Calixto and Medeiros. 55</p><p>Moreover, among the phenolic and flavonoid compounds identified in</p><p>EHFLR, quercetin, rutin, chlorogenic acid, caffeic acid and gallic acid</p><p>have already been reported to have anti-inflammatory activity in dif-</p><p>ferent inflammation models, acting in different inflammatory cascade</p><p>stages. 56–59</p><p>5. Conclusions</p><p>Our results demonstrated that the hydroalcoholic extract of the</p><p>leaves of Licania rigida Benth (EHFLR), rich in phenolic compounds and</p><p>flavonoids, presented an anti-inflammatory effect in models of systemic</p><p>inflammation, being possibly acting inhibiting the action of vasoactive</p><p>amines, besides probably intervening in the route of metabolism of</p><p>arachidonic acid. The extract also showed a reduction in leukocyte</p><p>migration, indicating that its action may be linked to the inhibition of</p><p>cytokine production. These results can be indicate that EHFLR as im-</p><p>portant anti-inflammatory with pharmacological efficacy.</p><p>Research funding</p><p>Cearense Foundation for Scientific and Technological Development</p><p>Support (FUNCAP).</p><p>Conflict of interest</p><p>Authors declare no conflict of interest.</p><p>References</p><p>1. Silva FOC, Macedo DV. Exercício físico, processo inflamatório e adaptação: uma visão</p><p>geral. 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