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Article Cite This: ACS Omega 2019, 4, 1221−1227
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Preclinical Evaluation of Rutin-Loaded Microparticles with an Enhanced Analgesic Effect Daniela Cristina de Medeiros,† Sandra Satie Mizokami,† Natalia Sfeir,† Sandra Regina Georgetti,‡ Alexandre Urbano,§ Rubia Casagrande,‡ Waldiceu A. Verri,†,∥ and Marcela Maria Baracat*,‡,∥ †
ACS Omega 2019.4:1221-1227. Downloaded from pubs.acs.org by 193.93.194.131 on 01/16/19. For personal use only.
Department of Pathological Sciences, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid Km 480 PR 445, CEP 86057-970 Londrina, Paraná, Brazil ‡ Department of Pharmaceutical Science, Universidade Estadual de Londrina, Avenida Robert Koch, 60, CEP 86038-350 Londrina, Paraná, Brazil § Department of Physics, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid Km 480 PR 445, CEP 86057-970 Londrina, Paraná, Brazil
ABSTRACT: This study aimed to develop and characterize microparticles containing rutin to improve the analgesic activity of the flavonoid. Rutin-loaded microparticles were produced with casein and pectin using the complex coacervation physicochemical method, resulting in an average particle size of 4.903 μm ± 4.421 (mean ± standard deviation), round shape, and irregular surfaces, and rutin crystals can be observed to be adsorbed on the outer surface of microparticles. The encapsulation efficiency was 76.9% as quantified by the antioxidant activity. In vivo, rutin-loaded microparticles showed greater inhibition of carrageenan-induced mechanical hyperalgesia (64%) than nonmicroencapsulated rutin (28%). The X-ray diffraction showed that rutin was dispersed in an amorphous matrix, and its crystallographic structure and crystal size did not exhibit changes. Differential scanning calorimetric studies confirmed that rutin was dispersed in the amorphous matrix within microparticles. The fact that rutin was dispersed in an amorphous matrix in the microparticles seemed to provide enhanced absorption, resulting in an improved analgesic efficacy compared with non-microencapsulated rutin. In conclusion, rutin-loaded microparticles were successfully produced, and they improved analgesic activity compared to non-microencapsulated rutin.
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INTRODUCTION Drug delivery systems are used to transport pharmaceutical compounds to specific localizations in order to exert their therapeutic effect. Current drug delivery systems have been developed with the aim of improving pharmacokinetics and clinical profiles, as well as the patient adherence to the treatment.1 Phytochemicals with low aqueous solubility present challenges for the development of formulations. One approach to overcome this difficulty is the encapsulation of such compounds, improving the dispersibility in water and consequently the bioavailability and biological activity.2 Microencapsulation by complex coacervation consists of a phase separation process using oppositely charged polymers. Microparticles then form via coacervate formation and deposition on the active substance.3,4 The composition of the polymers employed, solubility of the compounds, and pH of reactional media, among other factors, influence the final product.5 The advantages of this process include good control © 2019 American Chemical Society
of release, easy preparation, high resistance, and high encapsulation efficiency.6 Rutin is a flavonoid found in many dietary sources such as onion, grape, apple, and tomato and drinks such as wine and black tea.7 Of the pharmacological activities attributed to this compound, the antibacterial,8 antiviral,9 antiprotozoal,10 antitumor,11 antiallergic,12 anti-inflammatory,13 antiulcer,14 anti-oxidation,15 anti-diabetes,16 myocardial protecting,17 vasodilator,18 immunomodulator,19 and cognitive impairment prevention activities are clinically relevant.20,21 These studies highlight the potential of rutin in therapeutic applications.22 Rutin is poorly soluble in water, which reduces the oral bioavailability.23 Furthermore, studies have demonstrated that the in vivo effects of phenolic compounds such as rutin are dependent on absorption and metabolism in the gastroReceived: October 18, 2018 Accepted: January 8, 2019 Published: January 15, 2019 1221
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Particle size distribution (PSD) analysis was performed using light scattering. For removing the particles adhered in the polymer plate, the samples were dispersed in water and shaken in an electronic stirrer (vortex). The PSD of rutin-loaded microparticles revealed an average diameter of 4.903 μm ± 4.421 [mean ± standard deviation (SD)] (Figure 2). Ten percent (10%) of particles presented a diameter of ≤1.535 μm (d10 = 1.535 μm) and 90% of particles were ≤7.462 μm in diameter (d90 = 7.462 μm). Empty microparticles presented an average diameter of 19.15 μm ± 18.04 (mean ± SD) (data not shown), d10 of 2.098 μm, and d90 of 44.23 μm. Determination of Encapsulation Efficiency. The amount of rutin was determined by antioxidant activity determination by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A microencapsulation efficiency of 76.9% ± 6.3% (mean ± SD) was achieved. In Vitro Dissolution Studies. The in vitro dissolution profile of microencapsulated rutin in a medium that simulates the pH of gastric fluid (0−120 min) and of intestinal fluid (120−600 min) is shown in Figure 3. There was no release of rutin in the first 120 min of the dissolution test, during which time microparticles were exposed to the medium simulating gastric fluid. In the medium simulating intestinal fluid, a gradual release of rutin was observed, reaching 81.82% ± 9.58 (mean ± SD) by 10 h of assay. Differential Scanning Calorimetric Analysis. The differential scanning calorimetry (DSC) thermograms for non-microencapsulated rutin and rutin-loaded microparticles are presented in Figure 4. The main thermal events observed in the thermogram of the free rutin are indicated in the figure (132, 176, 214, and 235 °C). X-ray Diffraction Analysis. The X-ray diffraction patterns of non-microencapsulated rutin (Figure 5A) and rutin-loaded microparticles (Figure 5B) allowed the crystalline rutin phase to be identified as pattern diffraction file 00-005-044 in both samples using the HighScore Plus crystallographic database. Rutin-loaded microparticles exhibited reduced peak intensity compared with non-microencapsulated rutin because the amount of rutin differed between samples. The volume-averaged crystallite size (D) was measured using 5.30° and 26.83° (2θ) peaks which gave 59.51 ± 10.00 nm for pure rutin and 79.73 ± 10.00 nm for rutin-loaded microparticles. Evaluation of the Therapeutic Effect of Rutin-Loaded Microparticles in Carrageenan-Induced Mechanical Hyperalgesia in Mice. Carrageenan caused significant mechanical hyperalgesia in mice at all time points tested, peaking at 3 h compared with the control group (Figure 6). Empty microparticles did not alter the carrageenan-induced mechanical hyperalgesia. Non-microencapsulated rutin reduced carrageenan-induced mechanical hyperalgesia at 3, 5, and 7 h. Analgesic effects of rutin-loaded microparticles started at 0.5 h and continued until 7 h. The magnitude of this analgesic effect was significantly greater than that of nonmicroencapsulated rutin at 3, 5, and 7 h (Figure 6). At the maximum peak of carrageenan-induced mechanical hyperalgesia (3 h), rutin-loaded microparticles resulted in 64% of analgesia, while 28% was observed for non-microencapsulated rutin.
intestinal tract. Only 5−10% of ingested polyphenols are absorbed in the small intestine, and the colonic microbiota are responsible for the breakdown of 90−95% of these compounds into low molecular weight metabolites, which are key in the biological activities attributed to these compounds.24 Thus, the development of sustained-release formulations that pass through the gastrointestinal tract and release the compounds in the colon may improve the absorption of these substances. The development of sustained-release formulations of phenolic substances in the colon is therefore of interest. The aim of this study was to improve the therapeutic efficacy of rutin using microencapsulation. This paper describes the development of rutin-loaded microparticles to modify drug release and the morphological characteristics of the microparticles. Finally, the analgesic efficiency of the rutin-loaded microparticles in comparison with non-microencapsulated rutin in carrageenan-induced inflammatory pain in mice is reported.
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RESULTS Preparation of Microparticles and Morphological Analysis. Microparticles were prepared by complex coacervation with the dispersion of pectin, casein, and rutin in distilled water. The multiparticulate organized system was obtained by slow and gradual reduction of the pH (to 3.7 ± 0.1, below the pI of casein), performed under mild conditions (i.e., in the absence of organic solvents) (Figure 1).
Figure 1. Photomicrographs of microparticles obtained by scanning electron microscopy at a magnification of 2500× and 3600×. (A,B) Empty microparticles. (C,D) Rutin-loaded microparticles.
Microparticles were analyzed using scanning electron microscope, and the morphology was evaluated from the photomicrographs obtained at 2500× (Figure 1A,C) and 3600× (Figure 1B,D) magnification. Both empty microparticles (Figure 1A,B) and rutin-loaded microparticles (Figure 1C,D) presented round shape and irregular surfaces, with particles adhered in polymeric plaque. The photomicrographs of rutin-loaded microparticles reveal the presence of rutin adsorbed on the outer surface of microparticles, in a crystal shape (Figure 1C,D).
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DISCUSSION The main goal of drug delivery research is to develop formulations that fulfill the therapeutic needs of particular 1222
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Figure 2. PSD of rutin-loaded microparticles.
Figure 3. In vitro dissolution profile of microencapsulated rutin in a medium that simulates gastric medium (0−120 min) and enteric medium (120−600 min). In all cases, mean values (n = 6) are presented.
the same polymer (pectin/casein) but dried by spray drying did not form agglomerated particles.34 Factors that influence the size distribution of microparticles include the core-to-wall ratio, stirring speed of the preparation process,35 drying process,27 drug/polymer ratio,36 and homogenization cycles.37 The average diameter of rutin-loaded microparticles was 4.903 μm ± 4.421 in the present study. Additionally, the d10 was 1.535 μm, which means that 10% of particles had a diameter ≤1.535 μm; and the d90 was 7.462 μm; therefore, 80% of the particles were 1.535−7.462 μm in diameter. Empty microparticles had an average diameter of 19.15 μm ± 18.04, d10 of 2.098 μm, and d90 of 44.23 μm, which means that 80% of particles were 2.098−44.23 μm. The standard deviation observed in the analysis of empty microparticles was larger than that observed for rutin-loaded microparticles, which may be related to the presence of the drug. In support of this suggestion, an increase of homogeneity was also observed in acetaminophen-loaded microparticles compared with formulations without acetaminophen.28 In addition to the morphology and diameter, microencapsulation efficiency is also an important parameter. The microencapsulation efficiency of rutin-loaded microparticles
pathological conditions. Microparticulate systems can modify and control drug release, even when administrated by clinically important routes such as parental and peroral. These microparticulate systems can be either biodegradable or nonbiodegradable.27 The process of coacervation for the production of microparticles has been used in many studies.28−31 For instance, there are reports on the microencapsulation of sulfamerazine by pectin/gelatin complex coacervates,31 and we have previously applied the methodology to obtain microparticles containing acetaminophen28 and quercetin.32 In the present study, the complex coacervation method was successfully applied to generate pectin/casein microparticles containing rutin. Rutin-loaded microparticles were observed to present round shape and irregular surfaces, with clusters of microparticles and drug crystals attached and/or adsorbed on the external surface of the microparticles. Changing the production parameters leads to the formation of microparticles with different morphological characteristics.33 The drying process can affect the coalescence of particles, and microparticles prepared using 1223
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have obtained encapsulation efficiencies of 89% for methotrexate in microparticles prepared with biocompatible hyaluronic acid and sodium alginate.38 A similar result was reported for amoxicillin-loaded microparticles prepared by ionotropic gelation of sodium alginate with chitosan in the presence of calcium chloride, which presented a drug entrapment efficiency of 84%.36 In contrast, the encapsulation efficiency obtained for rutin in nanoparticles of sodium caseinate and pectin has been reported to be 33.43% in acidified medium (pH 3.7) and 54.82% with thermal treatment.39 Therefore, our results present enhanced rutin microencapsulation efficiency compared with previous studies.39 The release of rutin in medium simulating intestinal fluid was gradual, reaching 81.82% after 10 h. The lack of rutin release in the medium simulating gastric fluid is likely due to the low solubility of rutin in acid and the protection conferred by the extensive hydrolysis of casein/pectin complex formation in these pH conditions.34 Supporting this, alkaline conditions can cause deprotonation of the phenolic group of rutin, increasing its solubility.2 The prolonged release of rutin from microparticles could indicate that delayed release would occur in the large intestine because of degradation of the pectin/ casein microparticles by intestinal pectinase32 and could be responsible for the improvement in the analgesic effect observed in this study. In the DSC analyses, the first endothermic peak (132 °C) observed in the thermograms of free rutin can be characterized by dehydration. The endothermic peak of phase transition observed at 176 °C indicates a change in sample behavior related to molecular rearrangement of the rutin polymorphic state in a plastic substance.25 The peak at 214 °C suggests the occurrence of boiling, consistent with published data.25,26 This peak was not observed in the DSC curve of rutin-loaded microparticles, which indicates that the rutin is located inside of the microparticles. The X-ray diffraction experiments were performed to determine whether non-microencapsulated and rutin-loaded microparticles exhibited differences in their crystal structures and crystallite size. The presence of peaks at the same angular position for the pure drug indicated that the substance remained crystalline following microencapsulation. However, the halo observed in the inset of Figure 5 indicates the presence of amorphous material in microparticles, probably from pectin/casein polymers. The difference between the crystallite sizes of rutin powder and microencapsulated rutin was not significant, indicating that microencapsulation did not change the crystallographic characteristics of rutin substantially. Corroborating the results obtained in vitro, microencapsulation significantly increased the analgesic effect of rutin in the carrageenan-induced paw inflammation model. Rutin-loaded microparticles offered faster effects and increased analgesia compared to non-microencapsulated rutin. Microencapsulation of quercetin also improved its antiinflammatory effect in acetic acid-induced colitis, which was attributed to the local delivery of quercetin in the large intestine due to colonic microbiota action.32 Microencapsulation can also increase the total time of effect; for instance, an increase in the efficacy and extension of the local anesthetic effect of bupivacaine has been observed after subcutaneous infiltration when the drug is incorporated in biodegradable microparticles.40
Figure 4. Differential scanning calorimetric analysis thermograms for: (A) non-microencapsulated rutin, (B) rutin-loaded microparticles, and (C) empty microparticles.
Figure 5. X-ray diffraction patterns of (A) free rutin, (B) rutin-loaded microparticles, and (C) empty microparticles.
Figure 6. Microencapsulation of rutin increased its analgesic effects. Results are presented as mean ± standard error of the mean of six mice per group per experiment and are representative of two separate experiments. Analysis was carried out by one-way ANOVA followed by Bonferroni’s multiple comparison test. *P < 0.05 compared with the control group, #P < 0.05 compared with the untreated carrageenan group; **P < 0.05 compared with the group without carrageenan treatment and the non-microencapsulated rutin group.
was 76.9%, which is consistent with the literature and represents a high efficiency rate. For instance, other researchers 1224
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Texas, USA); and rutin from Acrós Organics (Geel, Antwerp, Belgium). Animals. Male Swiss mice (25−30 g) from the Universidade Estadual de Londrina, Londrina, Parana, Brazil, were used in this study. Mice were housed in standard clear plastic cages with free access to food and water, a light/dark cycle of 12:12 h, and controlled temperature. All behavioral testing was conducted during the light cycle in a temperaturecontrolled room. Animal care and handling procedures were approved by the Ethics Committee for Animal Use of the Universidade Estadual de Londrina under the process number 3324.2013.58. All efforts were made to minimize the number of animals used and their suffering. Experiments were doubleblinded. Preparation of Microparticles. Microparticles were prepared by dispersion of pectin and casein in distilled water (solid content 10%, being 5% pectin and 5% casein, w/v) under constant mechanical shaking. Sodium hydroxide (4.0 M) was used to adjust the pH to 8.0 ± 0.1.28 After complete dispersion, rutin was added in a ratio of 1:5 (drug/polymer). Microparticles were obtained by slow and gradual reduction of the pH with 1.0 M citric acid until it reached 3.7 ± 0.1. The same methodology was used to prepare microparticles containing no drug (empty microparticles). The samples were dried in a lyophilizer (Edwards, model Pirani 501) for 24 h. Morphologic Characteristics. The microparticles were coated with gold/palladium under an argon atmosphere and examined using a scanning electron microscope (Quanta 200, FEI). The morphology was evaluated from photomicrographs obtained at 2500× magnification for empty and rutin-loaded microparticles. The analysis of PSD was performed by light scattering using a Zetasizer Nano system ZS (Malvern Instruments). Determination of Encapsulation Efficiency. To determine the encapsulation efficiency, 101.8 mg of microparticles was dispersed in 10 mL of phosphate buffer pH 6.8 with 5.0% Tween-20 and then stirred for 5 min, followed by centrifugation at 3600 rpm for 10 min. The supernatant was filtered through a 0.45 μm nylon syringe filter, and the filtrate analyzed using the DPPH method42,43 and quantified spectrophotometrically at 517 nm to determine drug content. Tween-20 was used because of the low solubility of the rutin in water. Concentrations of 1, 2.5, and 5% were tested (data not shown) being the 5% concentration that presented the best result. The analyses were performed in triplicate. Encapsulation efficiency was calculated using eq 1
The faster analgesic effect of rutin-loaded microparticles compared with non-microencapsulated rutin indicated that microencapsulation modified an additional factor to the release time, extending the analgesic effect. The presence of the amorphous phase due to pectin and casein polymers explains the increased analgesic effect of rutin-loaded microparticles compared with non-microencapsulated rutin. Differential scanning calorimetric and X-ray diffraction analyses confirmed that crystalline rutin was dispersed in the amorphous polymer matrix of microparticles. There has been an increase in the number of newly developed drug molecules with poor water solubility and bioavailability, rendering it necessary to improve the wettability and oral bioavailability of these novel drugs. Physical modifications can be used to improve the wettability of a drug, including the reduction of particle size and consequent increase of the surface area.41 However, in the case of compounds with very low solubility, a reduction in particle size is not sufficient for the compound to become soluble and achieve therapeutic concentrations. In these cases, amorphous phase generation is an alternative approach because amorphous substances have higher solubilities than crystalline ones.37 The fact that rutin was dispersed in an amorphous matrix in the microparticles seemed to provide enhanced absorption, resulting in an improved analgesic efficacy compared with nonmicroencapsulated rutin. Therefore, the faster analgesic effect of rutin-loaded microparticles may be dependent on binding to the amorphous phase of the microparticles, and the extended analgesic effect may be related to the later release of rutin in the large intestine as the pectin/casein complex degrades. The combination of both modifications may have contributed to the enhancement of the analgesic effect of rutin by microencapsulation.
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CONCLUSIONS The process described in the present study led to the successful production of rutin-loaded microparticles. Microparticles were predominantly spherical with uniform PSD. Analysis with X-ray diffraction and DSC confirmed that the drug was dispersed in an amorphous matrix and retained its original crystallographic structure. The presence of the amorphous phase of the polymers contributed to a faster analgesic effect, and delayed release in the large intestine resulted in an extended analgesic effect. The combination of both actions resulted in enhancement of the analgesic effect. To our knowledge, this is the first study reporting the development of a novel formulation to modify the release of rutin, with improvement of the in vivo action as assessed by the analgesic activity. Therefore, the production of rutin-loaded microparticles by complex coacervation presents a promising pharmaceutical approach to achieve improved analgesia of the flavonoid rutin.
% EE =
initial drug added − free drug × 100 initial drug added
(1)
where % EE is encapsulation efficiency. In Vitro Dissolution Studies. Dissolution studies were performed by measuring the percentage of rutin in solution at a predetermined sampling time. Drug release patterns were studied over 10 h using a dissolution tester (Erweka DT-6, USP Apparatus 1) at a rotational speed of 50 rpm and using 800 mg of microparticles. During the first 2 h, 500 mL of 0.1 M HCl (pH 1.2 ± 0.1, 37 °C) with 5.0% Tween-20 was used to simulate gastric fluid conditions. Then, 500 mL of monobasic potassium phosphate buffer (KH2PO4) with 5.0% Tween-20 (pH 6.8 ± 0.1) was added to simulate intestinal fluid conditions, and the test continued for another 8 h. Tween-
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EXPERIMENTAL SECTION Materials. Pectin USP (68% degree of esterification) was obtained from CPKelco (Limeira, SP, Brazil); casein from Katuffman & Co (Kehl, Baden-Württemberg, Germany); citric acid, sodium hydroxide, chloride acid, and potassium phosphate from Merck (analytical grade, Darmstadt, Hessen, Germany); Tween-20 from Synth (Diadema, SP, Brazil); carrageenan from Santa Cruz Biotechnology Inc. (Dallas, 1225
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zero-time mean measurements. Data are presented as mean ± standard error of the mean of six mice per group per experiment, and experiments were performed in duplicate. Statistical Analysis. Data were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni’s multiple comparisons test. Differences were considered significant when P < 0.05. Statistical analyses were performed using GraphPad Prism 4.0 software.
20 was used due of the low solubility of the rutin in water. Concentrations of 1, 2.5, and 5% were tested (data not shown) being the 5% concentration that presented the best result. The experiment was carried out six times, and the results expressed as mean ± SD. At the indicated time points, samples were collected and immediately filtered through 0.45 μm Millipore filter paper. Each sample was analyzed using the DPPH method, and the absorbance at 517 nm was measured. DSC Analysis. The DSC analysis was performed on rutin, empty microparticles, and rutin-loaded microparticles. The samples were heated from 30 to 500 °C at a heating rate of 5 °C/min under a nitrogen atmosphere using a Shimadzu DSC60. X-ray Diffraction Analysis. The X-ray diffraction experiments were performed in a Bruker D8 powder diffractometer using the Bragg−Brentano geometry in continuous mode with a scan speed of 0.05°/s. A Cu Kα (λ = 1.5405 Å) line focus radiation tube was operated at 40 kV and 30 mA, and 2θ angles were measured from 3 to 40°. Pure crystalline rutin and rutinloaded microparticle phases were identified using X’Pert HighScore Plus with the PDF2DB analytical database. The Scherrer equation (eq 2) was used to calculate the crystallite size for both samples, taking βinstrumental from the silicon standard as 0.07° (0.0012 rad), using a Lorentzian function to fit the peak and considering the form factor as k = 0.9.44−47 The β2θ value was obtained by subtracting the full width at half maximum of the sample (βsample) from that of the instrument (βinstrumental), as shown in eq 3.
D=
kλ β2θ cos θ
β2θ = βs − βi
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (M.M.B.). ORCID
Rubia Casagrande: 0000-0002-2296-1668 Waldiceu A. Verri: 0000-0003-2756-9283 Marcela Maria Baracat: 0000-0002-7109-7191 Author Contributions ∥
W.A.V. and M.M.B. share senior authorship.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Brazilian Grants from Conselho ́ Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Coordenaçaõ de Aperfeiçoamento de Pessoal de ́ Superior (CAPES), Ministério da Ciência Tecnologia e Nivel Inovaçaõ (MCTI), Secretaria da Ciência, Tecnologia e Ensino Superior (SETI), Fundação Araucária and Parana State Government.
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REFERENCES
(1) Goole, J.; Amighi, K. 3D printing in pharmaceutics: a new tool for designing customized drug delivery systems. Int. J. Pharm. 2016, 499, 376−394. (2) Pan, K.; Luo, Y.; Gan, Y.; Baek, S. J.; Zhong, Q. pH-driven encapsulation of curcumin in self-assembled casein nanoparticles for enhanced dispersibility and bioactivity. Soft Matter 2014, 10, 6820− 6830. (3) Gouin, S. Microencapsulation. Trends Food Sci. Technol. 2004, 15, 330−347. (4) Xiao, Z.; Liu, W.; Zhu, G.; Zhou, R.; Niu, Y. A review of the preparation and application of flavour and essential oils microcapsules based on complex coacervation technology. J. Sci. Food Agric. 2013, 94, 1482−1494. (5) Saravanan, M.; Rao, K. P. Pectin-gelatin and alginate-gelatin complex coacervation for controlled drug delivery: Influence of anionic polysaccharides and drugs being encapsulated on physicochemical properties of microcapsules. Carbohydr. Polym. 2010, 80, 808−816. (6) Onbas, R.; Kazan, A.; Nalbantsoy, A.; Yesil-Celiktas, O. Cytotoxic and Nitric Oxide Inhibition Activities of Propolis Extract along with Microencapsulation by Complex Coacervation. Plant Foods Hum. Nutr. 2016, 71, 286−293. (7) Becho, J. R. M.; Machado, H.; Guerra, M. O. Rutina − Estrutura, Metabolismo e Potencial Farmacológico. Rev. Int. Est. Exp. 2009, 1, 21−25. (8) Pimentel, R. B. d. Q.; da Costa, C. A.; Albuquerque, P. M.; Junior, S. D. Antimicrobial activity and rutin identification of honey produced by the stingless bee Melipona compressipes manaosensis and commercial honey. BMC Complementary Altern. Med. 2013, 13, 151. (9) Ibrahim, A. K.; Youssef, A. I.; Arafa, A. S.; Ahmed, S. A. AntiH5N1 virus flavonoids fromCapparis sinaicaVeill. Nat. Prod. Res. 2013, 27, 2149−2153.
Evaluation of the Analgesic Effect of Rutin-Loaded Microparticles in Carrageenan-Induced Paw Inflammation in Mice. The analgesic effects of rutin, empty microparticles, and rutin-loaded microparticles were compared in the carrageenan-induced paw inflammation in mice. Mice were treated with rutin (100 mg/kg, peroral), empty microparticles (equivalent to the amount of rutin-loaded microparticles), or rutin-loaded microparticles (100 mg of rutin/kg, peroral) 1 h before intraplantar (i.pl.; subcutaneous injection in the plantar face of hind paw) injection of carrageenan solution (300 μg) in saline (25 μL). The negative control received i.pl. injection of saline (25 μL). The mechanical hyperalgesia (increased sensitivity to painful mechanical stimulus) was evaluated by an electronic version of von Frey filaments as previously described in detail.48 Briefly, in a quiet room, mice were placed in acrylic cages (12 × 10 × 17 cm) with wire grid floors, 15−30 min before the start of testing. The test consisted of evoking a hind paw flexion reflex with a hand-held force transducer (electronic aesthesiometer; Insight) adapted with a 0.5 mm2 polypropylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the hind paw with a gradual increase in pressure. The end point was characterized by the removal of the paw followed by clear flinching movements. After paw withdrawal, the intensity of the pressure was recorded automatically. The value for the response was defined as the average of three measurements. The animals were tested before and after treatment. Results are expressed by delta (Δ) withdrawal threshold (in gram) calculated by subtracting the mean measurements at 0.5, 1, 3, 5, and 7 h after stimulus from the 1226
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DOI: 10.1021/acsomega.8b02868 ACS Omega 2019, 4, 1221−1227
