Acessibilidade / Reportar erro

Improved antimutagenic effect of Pyrostegia venusta (Ker Gawl.) Miers nanostructured extract in liposome and polymeric nanoparticle

Abstract

Pyrostegia venusta (Ker Gawl.) Miers, popularly known as “Cipó-de São-João”, has been used in traditional medicine for its therapeutic properties. Nanotechnology is able to enhance the pharmacological activity of plant extracts. In this context, liposomes and polymeric nanoparticles containing P. venusta ethanolic extract were developed and then physico-chemically characterized to evaluate the mutagenic/antimutagenic effects of P. venusta. In addition, transaminases and serum creatinine were biochemically analyzed for liver and renal damage, respectively. The micronucleus test was performed with male Swiss mice treated orally for 15 consecutive days with free extracts and nanostructured with P. venusta, and then intraperitoneally with N-ethyl-N-nitrosurea (50 mg/kg) on the 15th day of treatment. Micronucleated polychromatic erythrocytes (MNPCE) were evaluated in bone marrow. There was a significant reduction in the frequency of MNPCE (LPEPV = 183% and NPEPV = 114%, p < 0.001), indicating antimutagenic potential of the nanostructured extracts with P. venusta. The groups treated with only nanostructured extract did not show an increase in MNPCE frequency, and biochemical analyzes showed no significant difference between treatments. The liposomes and polymeric nanoparticles containing Pyrostegia venusta ethanolic extract showed biological potential in preventing the first step of carcinogenesis under the experimental conditions.

Keywords:
Pyrostegia venusta; Flavonoids; Nanotechnology; Micronucleus test

INTRODUCTION

Several toxic compounds have affinity with the organism and high capacity to react with our genetic material. When cells are exposed to a potentially toxic chemical, it is possible to then find small intracytoplasmic masses in the cell chromatin. These masses are found as a small cell nucleus in the cytoplasm outside the bigger nucleus, denominated the micronucleus (Queiroz et al., 2013Queiroz FM, Matias KWO, Cunha M.F, Schwarz A. Evaluation of (anti)genotoxic activities of Phyllanthus niruri L. in rat bone marrow using the micronucleus test. Braz J Pharm Sci. 2013;49(1):135-148.).

The formation of this micronucleus may be spontaneous, however some compounds have the ability to intensify the incidence of lesions, and it can characterize a mutagenic event for cells and possible apoptosis avoidance (Kirkland et al., 2005Kirkland D, Aardema M, Henderson L, Müller L. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity. Mutat Res Genet Toxicol Environ Mutagen. 2005;584(1-2):1-256.). Therefore, chemoprevention is based on the use of natural or synthetic agents to reverse, prevent or suppress the carcinogenic progression (Oliveira, Aldrighi, Rinaldi, 2006Oliveira VM, Aldrighi JM, Rinaldi JF. Quimioprevenção do câncer de mama. Rev Assoc Med Bras. 2006;52(6):453-459.). Thus, compounds with chemopreventive activity which occur in nature and can be obtained easily are of great relevance for public health and an alternative for reducing neoplasia rates (Huang, Plass, Gerhauser, 2011Huang J, Plass C, Gerhauser C. Cancer chemoprevention by targeting the epigenome. Curr Drug Targets. 2011;12(13):1925-1956.).

In this context, identifying chemopreventive agents is relevant as a possible strategy for cancer prevention. Some plants have demonstrated important chemopreventive and antineoplastic effects, but their use still has to be very careful, since many of them also present side effects (Kirkland et al., 2005Kirkland D, Aardema M, Henderson L, Müller L. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity. Mutat Res Genet Toxicol Environ Mutagen. 2005;584(1-2):1-256.; Queiroz et al., 2013Queiroz FM, Matias KWO, Cunha M.F, Schwarz A. Evaluation of (anti)genotoxic activities of Phyllanthus niruri L. in rat bone marrow using the micronucleus test. Braz J Pharm Sci. 2013;49(1):135-148.).

Therefore, developing studies which enable better understanding about the use of these agents and their possible pharmacological activity, either physiologically or molecularly, can elucidate a better risk/benefit analysis and selection against diseases related to genotoxic events (Kirkland et al., 2005Kirkland D, Aardema M, Henderson L, Müller L. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity. Mutat Res Genet Toxicol Environ Mutagen. 2005;584(1-2):1-256.; Queiroz et al., 2013Queiroz FM, Matias KWO, Cunha M.F, Schwarz A. Evaluation of (anti)genotoxic activities of Phyllanthus niruri L. in rat bone marrow using the micronucleus test. Braz J Pharm Sci. 2013;49(1):135-148.).

Pyrostegia venusta (Ker Gawl.) Miers belongs to the Bignoneaceae family, also known as Pyrostegia ígnea, or by its popular names of Cipó or Flor de São-João, and presents itself as an important natural source of antioxidants since it has a significant amount of secondary metabolites such as phenolic compounds which can behave as free radical inhibitors or suppressors (Roy et al., 2011Roy P, Amdekar S, Kumar A, Singh V. Preliminary study of the antioxidant properties of flowers and roots of Pyrostegia venusta (Ker Gawl) Miers. BMC Complement Altern Med. 2011;11:69-77.; Altoé et al., 2014Altoé TDP, Amorim GM, Gomes JVD, Borges AS, Valadão IC, Silva IV, et al. In vitro antioxidant and cell viability of Pyrostegia venusta (Ker Gawl.) Miers). Orbital: Electron J Chem. 2014;6(4):215-222.; Pereira et al., 2014Pereira AM, Hernandes C, Pereira SI, Bertoni BW, França SC, Pereira PS, et al. Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers. Chem Biol Interact. 2014;224:136-41.). Studies show that leaf and flower ethanolic extracts are used in traditional medicine to treat white spots on the body, known as leucoderma or vitiligo (Magalhães et al., 2010Magalhães EA, Júnior GJS, Campos TA, Silva LP, Silva RMG. Avaliação do potencial genotóxico do extrato bruto de Pyrostegia venusta (Ker Gawl.) Miers, Bignoneaceae, em medula óssea de camundongos. Rev Bras Farmacogn . 2010;20(1):65-69.).

Thus, nanotechnology is linked to the use of medicinal plants with relevant antioxidant activity, and is a science which develops technological products in nanometric scale. This connection aims to optimize therapeutic doses of vegetable compounds in stable physico-chemical structure with biological affinity. An increase in the therapeutic index, stability improvement, protection against physical and chemical degradation and maintenance of serum levels of nanostructured compounds have also been observed (Ourique et al., 2014Ourique AF, Charles PS, Souto GD, Pohlmann AR, Guterres SS, Beck RCR. Redispersible liposomal-N-acetylcysteine powder for pulmonary administration: development, in vitro characterization and antioxidant activity. ‎ Eur J Pharm Sci. 2014;65:174-182.). Plant extract matrices are complex and their tissue solubility and compatibility may be difficult. Thus, administration of these nanosystems is an effective alternative to reverse these factors (Lima, Albuquerque, 2012Lima ML, Albuquerque CN. Preparo e caracterização de nanopartículas de poli(ε -caprolactona) contendo megazol, agente anti-chagásico. Rev Bras Cienc Farm . 2012;93:493-498.).

Among these drug nanocarriers, liposomes and polymeric nanoparticles are important types of carriers used in research. Liposomes provide a broad range of size varying from nanometers to micrometers. They are constituted by one or more concentric phospholipidic bilayers with dispersed amphiphilic characteristics (Ajazuddin, 2010Ajazuddin SS. Applications of novel drug delivery system for herbal formulations. Fitoterapia. 2010;81(7):680-689.; Pereira et al., 2014Pereira AM, Hernandes C, Pereira SI, Bertoni BW, França SC, Pereira PS, et al. Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers. Chem Biol Interact. 2014;224:136-41.). Polymeric nanoparticles are mostly constituted by biodegradable and biocompatible synthetic polymers such as poly (ε-caprolactone) (PCL). These nanosystems containing bioactive compounds have been shown to have advantages because they act in disease/illness prevention or health promotion, promoting higher quality and safety for their use (Bitencourt et al., 2016Bitencourt PER, Ferreira LM, Cargnelutti LO, Denardi D, Boligon A, Fleck M, et al. A new biodegradable polymeric nanoparticle formulation containing Syzygium cumini: Phytochemical profile, antioxidant and antifungal activity and in vivo toxicity. Ind Crops Prod. 2016;83:400-407.).

In this context, the current study identified the presence of flavonoids in P. venusta leaf extract, evaluated the chemopreventive effect of the extract nanocarried in liposomes and polymeric nanoparticles on the damages induced to the DNA in vivo, as well as verified the possible hepatotoxic and nephrotoxic effects through hepatic transaminases (AST and ALT) and creatinine (CR) dosing, respectively.

MATERIAL AND METHODS

Animals

This research study was approved by the Ethics Committee on Animal Use (CEUA - UFMT/Cuiabá Campus, MT) (Protocol number: 23108.720740/2016-39), which is affiliated to the Council for Control of Animal Experiments (CONCEA). Male Swiss mice aged 6-7 weeks (weighing 25-30 g) were obtained from the breeding colonies of Central Biological Unit of the UFMT, Cuiabá campus. The mice were kept in plastic cages in an experimental room of LiPeQ/UFMT/Sinop-MT during the experimental period, under controlled temperature (22 ± 2 ºC), relative humidity (55 ± 10%), light cycle (12 hours light/dark), exhaust system conditions and fed with pelleted feed diet and filtered water ad libitum.

Chemicals

N-ethyl-N-nitrosourea (ENU, Sigma Aldrich, Saint Louis, USA), polysorbate 80 (Delaware, Porto Alegre, Brazil), poly (ε-caprolactone) with molecular weight (Mn) = 80.000 g/mol (PCL), sorbitan monooleate (Sigma-Aldrich, Lesquin, France), lipoid S75® (Sanbio Científica, São Paulo, Brazil) and caprylic/capric triglyceride (Brasquim, São Paulo, Brazil) were used in the experiment.

Pyrostegia venusta ethanolic extract

Pyrostegia venusta was collected in Sinop/MT at 11º52’12.8”S/55°31’18.4”W geographical coordinates in November of 2014, identified and stored at Herbário Centro-Norte Mato-Grossense (Voucher specimen CNMT 6207), UFMT/Sinop. The P. venusta extract was obtained from its leaves by drying and grinding them until obtaining a final mass of 2.39 kg. This material was macerated in 21.5 L of ethanol for 7 days; afterwards, the obtained extract was filtered and concentrated by rotary evaporator. The leaf ethanolic extract exhibited a yield of 5.6% with a mass of 135.7 g.

The flavonoids in the P. venusta leaf ethanolic extract were identified according to Duan et al. (2011Duan K, Yuan Z, Guo W, Meng Y, Cui Y, Kong D, et al. LC-MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract. J Ethnopharmacol. 2011;135(2):201-208.) with modifications. First, a comparison between the retention time of the sample being analyzed and the authentic external standards prepared in methanol was performed to characterize these flavonoids. The standards used were: amentoflavone, apigenin, kaempferol, luteolin, myricetin, quercetin, quercetin-3-β-d-glycoside, rutin and taxifolin (Sigma-Aldrich). The analysis was conducted by high-performance liquid chromatography/Tandem mass spectrometry (LC-MS/MS) by UPLC Agilent 1290 Infinity (Agilent Technologies, USA), Agilent Eclipse AAA column (4.6 x 150 mm, 3.5μm) at 25 ºC, and 20 μL of injection volume. Mobile phase gradient with acidified water by formic acid 0.1% (m/v) and acetonitrile in 05:95 to 95:05 (ACN: H2O) conditions. The run time was 33 min with a flow of 0.5 mL/min. Detection was obtained through mass spectrometry by Agilent 6460 Triple Quad with electrospray as ionization source and using nitrogen gas in the following conditions: gas temperature 300 ºC; flow 5 L/min; nebulizer pressure 45 psi; sheath gas temperature 250 ºC; flow 11 L/min; capillary - 3500 V and m/z scanning interval 120-900 units.

Preparation of nanostructures

The liposomes were developed by the reverse phase evaporation method (Mertins et al., 2005Mertins, O, Sebben M, Pohlmann AR, Da Silveira NP. Production of soybean phosphatidylcholine-chitosan nanovesicles by reverse phase evaporation: a step by step study. Chem Phys Lipids. 2005;138(1-2):29-37.). An aqueous phase with 1 mg/mL P. venusta ethanolic extract (m/v) and 0.25% (m/v) polysorbate 80 was prepared in phosphate buffered saline pH 7.4 and an organic phase with soybean phosphatidilcholine (1.2 g) and cholesterol (0.06 g) in 40 mL of chloroform. An aliquot of the aqueous dispersion (4 mL) was added over the organic phase and sonicated for 5 min, obtaining inverted micelle dispersion. The organic solvent was removed at 25 ºC by evaporation under reduced pressure, resulting in an organogel. Next, the remaining aqueous solution (96 mL) was added to the organogel and the system was kept under stirring for 30 min. Vesicles suffered extrusion through 0.22 µm membrane. In addition to the liposomes containing P. venusta ethanolic extract (LPEPV), liposomes without active extract (LPBl) were also developed, as described above.

Polymeric nanoparticles were developed by the solvent emulsification/evaporation method described by Quintanar-Guerrero et al. (1998Quintanar-Guerrero D, Alleman E, Doelker E, Fessi H. Preparation and characterization of nanocapsules from preformfed polymers by new process based on emulsification-diffusion technique. Pharm Res. 1998;15(7):1056-1062.) and Bitencourt et al. (2016Bitencourt PER, Ferreira LM, Cargnelutti LO, Denardi D, Boligon A, Fleck M, et al. A new biodegradable polymeric nanoparticle formulation containing Syzygium cumini: Phytochemical profile, antioxidant and antifungal activity and in vivo toxicity. Ind Crops Prod. 2016;83:400-407.). First, 1 mg/mL (m/v) of Pyrostegia venusta leaf ethanolic extract was dissolved in the aqueous phase containing 1% (m/v) polysorbate 80. The organic phase (ethyl acetate) was prepared with 1% (m/v) PCL and 1% (m/v) sorbitan monooleate. Both phases were subjected to gentle stirring at 40 ºC individually. The aqueous phase was then added over the organic phase after 60 min, originating the primary emulsion. This emulsion was kept under vigorous magnetic stirring for 20 min and a second aqueous phase containing 2% (m/v) polysorbate 80 was subsequently added over the first emulsion and transferred to a high shear mixer (mini homogenizer, TECNAL, model TE-103) at 6000 rpm for 10 min. Lastly, a rotary evaporator eliminated the solvent, originating the formulation called NPEPV. Nanoparticles without the active extract (NPBl) were also prepared for comparison purposes, as described above.

All formulations were prepared in triplicate and kept under refrigeration at 8 °C protected from light.

Physicochemical characterization of the nanostructured formulations

The nanostructured for mulations were macroscopically characterized according to their homogeneity, color and appearance. Formulations were microscopically evaluated according to their equivalent sphere average diameter (d4.3) and particle size distribution (Span) by laser diffraction (Malvern Mastersizer® 3000), confirmed through determining the average cumulative diameter (Z-average) and polydispersity index (PDI) by photon correlation spectroscopy analysis (Nanosizer® Nanoseries, Malvern Instruments, United Kingdom) at 25 °C. The pH determination was conducted by pHmeter (Gehaka, PGH2000) previously calibrated with pH 4.0 and 7.0 standards by directly inserting them into the formulations. Zeta potential (ξ) was determined through sample electrophoretic mobility (Zetasizer® Nano-ZS model ZEN 3600, Malvern Instruments, United Kingdom). Trials were performed in triplicate and the results plotted as the average of three determinations.

Micronucleus test in vivo

The acquisition and preparation of the erythrocyte slides from the bone marrow for the micronucleus (MN) frequency evaluation followed the methodology by MacGregor et al. (1987MacGregor JT, Heddle JA, Hite M, Margolin BH, Ramel C, Salamone MF, et al. Guidelines for the conduct of micronucleus assay in mammalian bone marrow erythrocytes. Mutat Res. 1987;189(2):103-12.). A total of 1000 cells per animal were analyzed by light microscope, 1000 times magnification (immersion) for slides prepared and decoded in a blind test and duplicated for each animal.

The MN frequency reduction percentage was calculated according to Sugui et al. (2003Sugui MM, Alves de Lima PL, Delmanto RD, da Eira AF, Salvadori DM, Ribeiro LR. Antimutagenic effect of Lentinula edodes (BERK.) Pegler mushroom and possible variation among lineages. Food Chem Toxicol. 2003;41(4):555-60.), through the equation below:

% reduction = ( Frequency of MN in A ) ( Frequency of MN in B ) ( Frequency of MN in A ) ( Frequency of MN in C ) x 100

Where A represents the group treated with ENU (positive control), B represents the group treated with P. venusta plus ENU, and C represents the group treated with 0.9% NaCl (negative control).

Experimental design

The mice were divided into 9 groups of 6 animals each, as referred below:

Group 1 - Negative control. The mice were treated with LPBl via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with 0.9% NaCl (0.1 mL/10g p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 2 - Negative control. The mice were treated with NPBl via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with 0.9% NaCl (0.1 mL/10g p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 3 - Positive control. The mice were treated with LPBl via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with ENU (50 mg/kg p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 4 - Positive control. The mice were treated with NPBl via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with ENU (50 mg/kg p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 5 - Group treated with LPEPV (1 mg/mL) via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with ENU (50 mg/kg p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 6 - Group treated with NPEPV (1 mg/mL) via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with ENU (50 mg/kg p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 7 - Group treated with LPEPV (1 mg/mL) via gavage with administration of 0.3 mL during all of the trial. The mice were intraperitoneally treated with 0.9% NaCl (0.1 mL/10g p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 8 - Group treated with NPEPV (1 mg/mL) via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with 0.9% NaCl (0.1 mL/10g p.c) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Group 9 - Group only treated with free Pyrostegia venusta leaf extract (EPV, 1 mg/mL) via gavage with administration of 0.3 mL during all of the trial period. The mice were intraperitoneally treated with ENU (50 mg/kg p.c.) on the 15th day; the mice were sacrificed on the next day (16th day) by decapitation followed by collecting cells from the bone marrow.

Biochemical analysis

Serum levels for hepatic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, as well as creatinine (CRT) in order to assess the renal activity were measured by Cobas Integra 400 Plus (Roche) equipment through the chemiluminescence method.

Statistical analysis

The frequency of micronucleated cells in the different trial groups was compared by the Chi-squared test (Pereira, 1991Pereira CAB. Teste estatístico para comparar proporções em problemas de citogenética. In: Rabello-Gay MN, Rodrigues MALR, Montelleone-Neto R, editor. Mutagênese, teratogênese e carcinogênese: métodos e critérios de avaliação. Acervo Fundacentro. São Paulo: Sociedade Brasileira de Genética; 1991, p.113-121.). The Tukey test with p<0.05 being considered statistically significant was used for the biochemical analysis in the SISVAR statistical program.

RESULTS

The nanostructured formulations (LPEPV and NPEPV) had a homogeneous macroscopic appearance, milky aspect and opalescent and slightly greenish color, which were expected characteristics due to the P. venusta extract.

The LPBl and NPBl formulations had a whitish color. All formulations had blue reflex caused by the Brownian motion of the nanosystems in the aqueous medium.

Table I presents the values of photon correlation spectroscopy (PCS) analysis of three batches of each formulation, showing monomodal particle size distributions with z-average diameters between 203 ± 3.61 nm and 293 ± 6.85 nm. As the PCS analyzes were performed with 11 scans of 10 s each, it is possible to infer the kinetic stability of the colloids (Fiel et al., 2013Fiel LA, Adorne MD, Guterres SS, Netz PA, Pohlmann AR. Variable temperature multiple light scattering analysis to determine the enthalpic term of a reversible agglomeration in submicrometric colloidal formulations: A quick quantitative comparison of the relative physical stability. Colloids Surf A Physicochem Eng Asp. 2013;431:93-104.). The polydispersity index (PDI) was lower than 0.2 for all formulations, and the results are consistent with studies on nanosystems (Bitencourt et al., 2016Bitencourt PER, Ferreira LM, Cargnelutti LO, Denardi D, Boligon A, Fleck M, et al. A new biodegradable polymeric nanoparticle formulation containing Syzygium cumini: Phytochemical profile, antioxidant and antifungal activity and in vivo toxicity. Ind Crops Prod. 2016;83:400-407.; Frank et al., 2019Frank LA, Gazzi RP, de Andrade Mello P, Buffon A, Pohlmann AR, Guterres SS. Imiquimod-loaded nanocapsules improve cytotoxicity in cervical cancer cell line. Eur J Pharm Biopharm. 2019;136:9-17.). The nanometric diameters of the formulations was confirmed by laser diffraction which demonstrated low polydispersion demonstrated by the range values, which indicates homogeneity in the samples. Zeta potential showed the presence of the surface charge potential of the nanosystems by electrophoretic mobility through the steric blockage of the nanostructures, with ξ near zero. A significant difference was observed in the potential comparison between NP and LP formulations, being considered very distinct formulations. Negative values are due to functional groups which contain oxygen present on the colloid surface and consequently have negative partial density at the pseudo-phase interface. The pH of LPBl and LPEPV was neutral and NPBl and NPEPV more acid, consistent with the composition of the formulations.

TABLE I
Nanostructure characterization along 30 days of study

There were five flavonoids (apigenin, luteolin, myricetin quercetin 3-β-d-glycoside and rutin) identified by LC-MS/MS in the P. venusta ethanolic extract (Figure 1) and demonstrated in Table II below.

FIGURE 1
Structures of the flavonoids identified in ethanolic extract from P. venusta.

TABLE II
LC-MS/MS parameters of the five flavonoids identified in the Pyrostegia venusta crude ethanolic extract

Table III presents the micronucleus frequency in polychromatic erythrocytes (PCEMNs) after pre-treatment with nanostructured and free P. venusta ethanolic extract. The results show 183% reduction in the frequency of PCEMNs for the LPEPV + ENU group (p<0.001) and 114% for the NPEPV + ENU group (p<0.001) if compared to their respective positive controls LPBl + ENU and NPBl + ENU, suggesting significant antimutagenic activity of the nanostructured extract, in addition to not showing a significant increase of PCEMNs if compared to the negative controls (LPBl + 0.9% NaCl and NPBl + 0.9% NaCl).

TABLE III
Frequency of micronuclei in polychromatic erythrocytes (MNPCEs) of bone marrow of mice after pre-treatment with Pyrostegia venusta ethanolic extract

Table IV presents the feed consumption (g/week/ group) and body weight (g) during the animal treatment period. The results suggest that the treatments did not cause toxicity to the animals, since there was no interference in the feeding nor to the weight gain.

TABLE IV
Feed average consumption (g/week/group) and body weight (g) of mice treated during 15 days with Pyrostegia venusta ethanolic extract

Table V presents transaminases (AST and ALT) and creatinine dosages regarding the possible toxicity in the administration of P. venusta ethanolic extract during the trial period. The results show that there was no significant difference among the treated groups, demonstrating that there were no hepatic or renal damage regarding P. venusta administration.

TABLE V
Biochemical analysis of the hepatic (AST and ALT) and renal (CRT) activities of control and treated with Pyrostegia venusta ethanolic extract groups during 15 days

DISCUSSION

The developed formulations showed a homogeneous macroscopic aspect of whitish color if formulated without P. venusta ethanolic extract (LPBl and NPBl). This result is in accordance with the appearance of the formulations and consistent with what is described in the literature for these types of nanostructured systems (Fiel et al., 2013Fiel LA, Adorne MD, Guterres SS, Netz PA, Pohlmann AR. Variable temperature multiple light scattering analysis to determine the enthalpic term of a reversible agglomeration in submicrometric colloidal formulations: A quick quantitative comparison of the relative physical stability. Colloids Surf A Physicochem Eng Asp. 2013;431:93-104.; Bittencourt et al., 2016Bitencourt PER, Ferreira LM, Cargnelutti LO, Denardi D, Boligon A, Fleck M, et al. A new biodegradable polymeric nanoparticle formulation containing Syzygium cumini: Phytochemical profile, antioxidant and antifungal activity and in vivo toxicity. Ind Crops Prod. 2016;83:400-407.). On the other hand, other formulations showed a milky greenish aspect after extract incorporation (LPEPV and NPEPV) and a fresh green leaf smell, characteristic of P. venusta ethanolic extract.

The suspension diameter evaluations demonstrated nanometer particle sizes between 200 and 300 nm. The systems presented low polydispersion as evaluated by Dynamic light scattering and confirmed by laser diffraction (Frank et al., 2019Frank LA, Gazzi RP, de Andrade Mello P, Buffon A, Pohlmann AR, Guterres SS. Imiquimod-loaded nanocapsules improve cytotoxicity in cervical cancer cell line. Eur J Pharm Biopharm. 2019;136:9-17.).

A cationic group in the formulation resulting from phosphatidylcholine provided a neutral pH to the liposomes (Perttu, Kohli, Szoka, 2012Perttu EK, Kohli AG, Szoka FC. Inverse-phosphocholine lipids: A remix of a common phospholipid. J Am Chem Soc. 2012;134(10):4485-4488.). On the other hand, the pH of the nanoparticles suspensions was in accordance with Ribeiro et al. (2016Ribeiro RF, Motta MH, Härter APG, Flores FC, Beck RCR., Schaffazick SR, et al. Spray-dried powders improve the controlled release of antifungal tioconazole-loaded polymeric nanocapsules compared to with lyophilized products. Mater Sci Eng C. 2016;59:875-884.), shown by the authors around pH 5.44. This NP characteristic is due to the presence of biodegradable polymer (PCL) with terminal carboxylic acid groups and therefore reduces the pH (Ribeiro et al. (2016)Ribeiro RF, Motta MH, Härter APG, Flores FC, Beck RCR., Schaffazick SR, et al. Spray-dried powders improve the controlled release of antifungal tioconazole-loaded polymeric nanocapsules compared to with lyophilized products. Mater Sci Eng C. 2016;59:875-884.. The ζ produces electrostatic repulsion among its neighbor colloids, being extremely relevant since it avoids agglomeration (Perttu, Kohli, Szoka, 2012Perttu EK, Kohli AG, Szoka FC. Inverse-phosphocholine lipids: A remix of a common phospholipid. J Am Chem Soc. 2012;134(10):4485-4488.).

The bioactive compounds from plant extracts show difficulty in intestinal absorption due to their low lipid solubility and the use of nanosystems improves this absorption by oral administration (v.o). These carriers enhance and optimize the ability of compounds such as flavonoids to enter and store within tumor tissues, improving the permeability effect and optimizing serum levels. In addition to enhancing the prevention of genetic damage, therapeutic doses of compounds extracted from plants are intensified through changes in their structures to improve their affinity in the body (Dong, Mumper, 2010Dong X, Mumper RJ. Nanomedicinal strategies to treat multidrug-resistant tumors: current progress. Nanomedicine (Lond). 2010;5(4):597-615.).

The results show that the use of nanosystems suggests an improvement in the bioavailability of compounds from P. venusta leaf extract and also demonstrates better biological activity by increasing the absorption of the components. A significant reduction in the frequency of micronucleus in polychromatic erythrocytes was observed for the mice treated with P. venusta nanostructured extract for both nanosystems used.

The higher potentiation of the antimutagenic effect of P. venusta occurred when carried in liposomes, as it is believed that nanocarriers improve the digestive system absorption, as well as improve bloodstream stability and metabolism of this extract (Dong, Mumper, 2010Dong X, Mumper RJ. Nanomedicinal strategies to treat multidrug-resistant tumors: current progress. Nanomedicine (Lond). 2010;5(4):597-615.). On the other hand, there are no studies of P. venusta related to its protective effect against genotoxic agents by using polymeric nanocapsules, making this study an original work about the identification of protective effect of nanostructured P. venusta for cancer prevention, suggesting an effective antioxidant activity of the flavonoids in this specie.

Both the liposome and polymeric nanoparticle nanosystems protect phytochemical compounds against obstacles such as physical and chemical degradation, biotransformation processes, withstanding the unexpected and abrupt degradation (Nair et al., 2010Nair HB, Sung B, Yadav VR, Kannappan R, Chaturvedi MM, Aggarwal BB. Delivery of antiinflammatory nutraceuticals by nanoparticles for the prevention and treatment of cancer. Biochem Pharmacol . 2010;80(12):1833-1843.). The advantage of using liposomes is mainly due to their characteristic phospholipidic bilayer, which improves the likely limitations of the poor bioavailability of flavonoids, for example. This turns their ability to avoid degradation into a perspective for their use in anticancer therapy (Lim, Lee, Kim, 2004Lim SJ, Lee MK, Kim CK. Altered chemical and biological activities of all-trans retinoic acid incorporated in solid lipid nanoparticle powders. J Control Release. 2004;100(1):53-61.). Moreover, their efficient protection is also related to the ability of liposomes of being captured by macrophages, increasing their concentration in liver, spleen and bone marrow (Frézard et al., 2005Frézard F, Schettini DA, Rocha OGF, Demicheli C. Lipossomas: Propriedades físico-químicas e farmacológicas, aplicações na quimioterapia à base de antimônio. Quim Nova. 2005;28(3):511-518.).

In accordance with Ferreira et al. (2009Ferreira FG, Regasini LO, Oliveira AM, Campos JADB, Silva DHS, Cavalheiro AJ, et al. Avaliação de mutagenicidade e antimutagenicidade de diferentes frações de Pterogyne nitens (leguminosae) utilizando ensaio de micronúcleo em Tradescantia pallida. Rev Bras Farmacogn. 2009;19(1a):61-67.), the chemopreventive activity over the DNA may be linked to flavonoids, which act as potent antimutagenic agents due to their antioxidant activity. In this context, the following flavonoids were identified in the P. venusta leaf ethanolic extract: apigenin (1), luteolin (2), myricetin (3), quercetin-3-β-d-glycoside (4) and rutin (5).

Quercetin has also been identified by Roy et al. (2011Roy P, Amdekar S, Kumar A, Singh V. Preliminary study of the antioxidant properties of flowers and roots of Pyrostegia venusta (Ker Gawl) Miers. BMC Complement Altern Med. 2011;11:69-77.) in P. venusta flowers and roots and by Veggi, Cavalcanti and Meireles (2014Veggi PC, Cavalcanti RN, Meireles MAA. Production of phenolic-rich extracts from Brazilian plants using supercritical and subcritical fluid extraction: Experimental data and economic evaluation. J Food Eng. 2014;131:96-109.) in leaves. Pereira et al. (2014Pereira AM, Hernandes C, Pereira SI, Bertoni BW, França SC, Pereira PS, et al. Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers. Chem Biol Interact. 2014;224:136-41.) found rutin in P. venuta flowers. Apigenin (1) and luteolin (2) were found in other species of the Bignoneaceae family, however this is their first report in P. venusta (Zoghbi, Oliveira, Guilhon, 2009Zoghbi MDGB, Oliveira J, Guilhon GMSP. The genus Mansoa (Bignoniaceae): A source of organosulfur compounds. Braz J Pharmacogn. 2009;19(3):795-804.). According to Blatt, Santos and Salatino (1998Blatt CTT, Santos MDD, Salatino A. Flavonoids of Bignoniaceae from the “ cerrado ” and their possible taxonomic significance. Plant Syst Evol. 1998;210:289-292.), rutin seems to be easily found in species cultivated in Brazilian cerrado, as these authors identified this flavonoid in P. venusta leaves. The identification of myricetin (3) in the Bignoneaceae family is also unprecedented (Pereira et al., 2014Pereira AM, Hernandes C, Pereira SI, Bertoni BW, França SC, Pereira PS, et al. Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers. Chem Biol Interact. 2014;224:136-41.).

The antioxidant activity of P. venusta leaves had been previously observed in in vitro tests by Altoé et al. (2014Altoé TDP, Amorim GM, Gomes JVD, Borges AS, Valadão IC, Silva IV, et al. In vitro antioxidant and cell viability of Pyrostegia venusta (Ker Gawl.) Miers). Orbital: Electron J Chem. 2014;6(4):215-222.). Pereira et al. (2014Pereira AM, Hernandes C, Pereira SI, Bertoni BW, França SC, Pereira PS, et al. Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers. Chem Biol Interact. 2014;224:136-41.) verified the antioxidant activity of phenolic compounds in P. venusta flower free extract. Khan, Afaq and Mukhtar (2008Khan N, Afaq F, Mukhta H. Cancer chemoprevention through dietary antioxidants: progress and promise. Antioxid Redox Signal. 2008;10(3):475-510.) reported that agents able to interfere in more than one crucial point in the carcinogenesis process may act in blocking the attachment of an injury in the DNA for cancer prevention, showing an advantage over the single-target agents.

It is known that quercetin acts on the cellular cycle regulation and is able to induce apoptosis in tumor cells, in addition to inhibiting tyrosine kinase activity (Dornas et al., 2007Dornas WC, Oliveira TT, Rodrigues-Das-Dores RG, Santos AF, Nagem TJ. Flavonóide: potencial terapêutico no estresse oxidativo. Rev Bras Cienc Farm. 2007;28(3):241-249.). Apigenin and quercetin act together by preventing cancer through a proteasome inhibition mechanism selective to cancer which does not affect normal cells, contributing to preventive effects of the disease (Chen et al., 2005Chen D, Daniel KG, Chen MS, Kuhn DJ, Landis-Piwowar KR, Dou QP. Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells. Biochem Pharmacol. 2005;69(10):1421-1432.). Luteolin adds action to the other flavonoids inducing the apoptosis of tumor cells by interrupting the cellular cycle in the G0/G1 phase, which may be the mechanism which luteolin uses to reduce the cellular viability of damaged cells (Cao et al., 2017Cao Z, Zhang H, Cai X, Fang W, Chai D, Wen Y, et al. Luteolin Promotes Cell Apoptosis by Induci ng Autophagy in Hepatocellular Carcinoma. Cell Physiol Biochem. 2017;43(5):1803-1812.). Apigenin has the ability to promote dissipation of absorbed energy, increasing its action level as antioxidant defense (Gobbo-Neto, Lopes, 2007Gobbo-Neto L, Lopes NP. Plantas medicinais: fatores de influência no conteúdo de metabólitos secundários. Quim Nova . 2007;30(2):374-381.).

Therefore, it can be suggested that the antimutagenic effect observed by P. venusta leaf ethanolic extract might be a result of the synergism among the plant compounds, with a mechanism of action which has not yet been elucidated.

Regarding the genotoxic evaluation, the LPEPV, NPEPV, LPBl, NPBl and EPV nanosystems did not show mutagenic effect. In studying free P. venusta flower extract, Magalhães et al. (2010Magalhães EA, Júnior GJS, Campos TA, Silva LP, Silva RMG. Avaliação do potencial genotóxico do extrato bruto de Pyrostegia venusta (Ker Gawl.) Miers, Bignoneaceae, em medula óssea de camundongos. Rev Bras Farmacogn . 2010;20(1):65-69.) evaluated the clastogenic effects in different concentrations (50, 100 and 200 mg/ kg p.c) in Swiss mice by micronucleus test and did not verify a significant increase in the average number of cells with micronucleus or chromosomal defects.

The administration of free or nanostructured P. venusta extract also did not show a physical sign of toxicity in animals during the trial period such as weight loss, lack of water ingestion or loss of appetite. The data shows a weight gain during treatment, which is considered normal (Spinelli et al., 2012Spinelli MO, Cruz RJ, Godoy CMS, Motta MC. Comparação dos parâmetros bioquímicos de camundongos criados em diferentes condições sanitárias. Sci Plena. 2012;8(2):1-8.).

Furthermore, no hepatotoxic and nephrotoxic effects were observed from the AST, ALT and CRT dosages, respectively. The use of inert nanocarriers containing P. venusta extract did not demonstrate possible hepatic damage, confirming the ability of nanostructures to protect against undesirable interactions (Lim, Lee, Kim, 2004Lim SJ, Lee MK, Kim CK. Altered chemical and biological activities of all-trans retinoic acid incorporated in solid lipid nanoparticle powders. J Control Release. 2004;100(1):53-61.; Spinelli et al., 2012Spinelli MO, Cruz RJ, Godoy CMS, Motta MC. Comparação dos parâmetros bioquímicos de camundongos criados em diferentes condições sanitárias. Sci Plena. 2012;8(2):1-8.).

Finally, according to Roy et al. (2011Roy P, Amdekar S, Kumar A, Singh V. Preliminary study of the antioxidant properties of flowers and roots of Pyrostegia venusta (Ker Gawl) Miers. BMC Complement Altern Med. 2011;11:69-77.) and Mostafa, El-Dahshan and Singab, (2013Mostafa NM, El-Dahshan O, Singab ANB. Pyrostegia venusta (Ker Gawl.) Miers: A botanical, pharmacological and phytochemical review. Med Aromat Plants. 2013;2(03):123.), P. venusta has compounds with great potential in traditional medicine and in the discovery of new products with pharmacological potential.

CONCLUSION

In conclusion, the developed nanostructures had nanometric size with monomodal characteristic and physico-chemical stability compatible with the literature and the observed effects. identified total of five flavonoids with potent antioxidant activity were identified in the P. venusta leaf ethanolic extract, suggesting that the extract contains compounds which act in the prevention or neutralization of DNA damage induced by the chemical agent in vivo. In this context, there was antimutagenic activity potentiation of P. venusta extract carried in liposomes and polymeric nanoparticles when compared with the activity in the free form by the micronucleus test and without possible harmful effects to the mice during the experimental period. Thus, under the trial conditions, nanostructured P. venusta leaf ethanolic extract, blank nanostructures and the free extract did not present possible mutagenic, hepatotoxic or nephrotoxic effects in the mice, showing great pharmacological potential for cancer chemoprevention.

ACKNOWLEDGEMENTS

This work was supported by the FAPEMAT - “Fundação de Amparo à Pesquisa do Estado de Mato Grosso”, Mato Grosso, Brazil.

REFERENCES

  • Altoé TDP, Amorim GM, Gomes JVD, Borges AS, Valadão IC, Silva IV, et al. In vitro antioxidant and cell viability of Pyrostegia venusta (Ker Gawl.) Miers). Orbital: Electron J Chem. 2014;6(4):215-222.
  • Ajazuddin SS. Applications of novel drug delivery system for herbal formulations. Fitoterapia. 2010;81(7):680-689.
  • Bitencourt PER, Ferreira LM, Cargnelutti LO, Denardi D, Boligon A, Fleck M, et al. A new biodegradable polymeric nanoparticle formulation containing Syzygium cumini: Phytochemical profile, antioxidant and antifungal activity and in vivo toxicity. Ind Crops Prod. 2016;83:400-407.
  • Blatt CTT, Santos MDD, Salatino A. Flavonoids of Bignoniaceae from the “ cerrado ” and their possible taxonomic significance. Plant Syst Evol. 1998;210:289-292.
  • Cao Z, Zhang H, Cai X, Fang W, Chai D, Wen Y, et al. Luteolin Promotes Cell Apoptosis by Induci ng Autophagy in Hepatocellular Carcinoma. Cell Physiol Biochem. 2017;43(5):1803-1812.
  • Chen D, Daniel KG, Chen MS, Kuhn DJ, Landis-Piwowar KR, Dou QP. Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells. Biochem Pharmacol. 2005;69(10):1421-1432.
  • Dornas WC, Oliveira TT, Rodrigues-Das-Dores RG, Santos AF, Nagem TJ. Flavonóide: potencial terapêutico no estresse oxidativo. Rev Bras Cienc Farm. 2007;28(3):241-249.
  • Dong X, Mumper RJ. Nanomedicinal strategies to treat multidrug-resistant tumors: current progress. Nanomedicine (Lond). 2010;5(4):597-615.
  • Duan K, Yuan Z, Guo W, Meng Y, Cui Y, Kong D, et al. LC-MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract. J Ethnopharmacol. 2011;135(2):201-208.
  • Ferreira FG, Regasini LO, Oliveira AM, Campos JADB, Silva DHS, Cavalheiro AJ, et al. Avaliação de mutagenicidade e antimutagenicidade de diferentes frações de Pterogyne nitens (leguminosae) utilizando ensaio de micronúcleo em Tradescantia pallida Rev Bras Farmacogn. 2009;19(1a):61-67.
  • Fiel LA, Adorne MD, Guterres SS, Netz PA, Pohlmann AR. Variable temperature multiple light scattering analysis to determine the enthalpic term of a reversible agglomeration in submicrometric colloidal formulations: A quick quantitative comparison of the relative physical stability. Colloids Surf A Physicochem Eng Asp. 2013;431:93-104.
  • Frank LA, Gazzi RP, de Andrade Mello P, Buffon A, Pohlmann AR, Guterres SS. Imiquimod-loaded nanocapsules improve cytotoxicity in cervical cancer cell line. Eur J Pharm Biopharm. 2019;136:9-17.
  • Frézard F, Schettini DA, Rocha OGF, Demicheli C. Lipossomas: Propriedades físico-químicas e farmacológicas, aplicações na quimioterapia à base de antimônio. Quim Nova. 2005;28(3):511-518.
  • Gobbo-Neto L, Lopes NP. Plantas medicinais: fatores de influência no conteúdo de metabólitos secundários. Quim Nova . 2007;30(2):374-381.
  • Huang J, Plass C, Gerhauser C. Cancer chemoprevention by targeting the epigenome. Curr Drug Targets. 2011;12(13):1925-1956.
  • Kirkland D, Aardema M, Henderson L, Müller L. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity. Mutat Res Genet Toxicol Environ Mutagen. 2005;584(1-2):1-256.
  • Khan N, Afaq F, Mukhta H. Cancer chemoprevention through dietary antioxidants: progress and promise. Antioxid Redox Signal. 2008;10(3):475-510.
  • Lima ML, Albuquerque CN. Preparo e caracterização de nanopartículas de poli(ε -caprolactona) contendo megazol, agente anti-chagásico. Rev Bras Cienc Farm . 2012;93:493-498.
  • Lim SJ, Lee MK, Kim CK. Altered chemical and biological activities of all-trans retinoic acid incorporated in solid lipid nanoparticle powders. J Control Release. 2004;100(1):53-61.
  • MacGregor JT, Heddle JA, Hite M, Margolin BH, Ramel C, Salamone MF, et al. Guidelines for the conduct of micronucleus assay in mammalian bone marrow erythrocytes. Mutat Res. 1987;189(2):103-12.
  • Magalhães EA, Júnior GJS, Campos TA, Silva LP, Silva RMG. Avaliação do potencial genotóxico do extrato bruto de Pyrostegia venusta (Ker Gawl.) Miers, Bignoneaceae, em medula óssea de camundongos. Rev Bras Farmacogn . 2010;20(1):65-69.
  • Mertins, O, Sebben M, Pohlmann AR, Da Silveira NP. Production of soybean phosphatidylcholine-chitosan nanovesicles by reverse phase evaporation: a step by step study. Chem Phys Lipids. 2005;138(1-2):29-37.
  • Mostafa NM, El-Dahshan O, Singab ANB. Pyrostegia venusta (Ker Gawl.) Miers: A botanical, pharmacological and phytochemical review. Med Aromat Plants. 2013;2(03):123.
  • Nair HB, Sung B, Yadav VR, Kannappan R, Chaturvedi MM, Aggarwal BB. Delivery of antiinflammatory nutraceuticals by nanoparticles for the prevention and treatment of cancer. Biochem Pharmacol . 2010;80(12):1833-1843.
  • Oliveira VM, Aldrighi JM, Rinaldi JF. Quimioprevenção do câncer de mama. Rev Assoc Med Bras. 2006;52(6):453-459.
  • Ourique AF, Charles PS, Souto GD, Pohlmann AR, Guterres SS, Beck RCR. Redispersible liposomal-N-acetylcysteine powder for pulmonary administration: development, in vitro characterization and antioxidant activity. ‎ Eur J Pharm Sci. 2014;65:174-182.
  • Pereira AM, Hernandes C, Pereira SI, Bertoni BW, França SC, Pereira PS, et al. Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers. Chem Biol Interact. 2014;224:136-41.
  • Pereira CAB. Teste estatístico para comparar proporções em problemas de citogenética. In: Rabello-Gay MN, Rodrigues MALR, Montelleone-Neto R, editor. Mutagênese, teratogênese e carcinogênese: métodos e critérios de avaliação. Acervo Fundacentro. São Paulo: Sociedade Brasileira de Genética; 1991, p.113-121.
  • Perttu EK, Kohli AG, Szoka FC. Inverse-phosphocholine lipids: A remix of a common phospholipid. J Am Chem Soc. 2012;134(10):4485-4488.
  • Queiroz FM, Matias KWO, Cunha M.F, Schwarz A. Evaluation of (anti)genotoxic activities of Phyllanthus niruri L. in rat bone marrow using the micronucleus test. Braz J Pharm Sci. 2013;49(1):135-148.
  • Quintanar-Guerrero D, Alleman E, Doelker E, Fessi H. Preparation and characterization of nanocapsules from preformfed polymers by new process based on emulsification-diffusion technique. Pharm Res. 1998;15(7):1056-1062.
  • Ribeiro RF, Motta MH, Härter APG, Flores FC, Beck RCR., Schaffazick SR, et al. Spray-dried powders improve the controlled release of antifungal tioconazole-loaded polymeric nanocapsules compared to with lyophilized products. Mater Sci Eng C. 2016;59:875-884.
  • Roy P, Amdekar S, Kumar A, Singh V. Preliminary study of the antioxidant properties of flowers and roots of Pyrostegia venusta (Ker Gawl) Miers. BMC Complement Altern Med. 2011;11:69-77.
  • Spinelli MO, Cruz RJ, Godoy CMS, Motta MC. Comparação dos parâmetros bioquímicos de camundongos criados em diferentes condições sanitárias. Sci Plena. 2012;8(2):1-8.
  • Sugui MM, Alves de Lima PL, Delmanto RD, da Eira AF, Salvadori DM, Ribeiro LR. Antimutagenic effect of Lentinula edodes (BERK.) Pegler mushroom and possible variation among lineages. Food Chem Toxicol. 2003;41(4):555-60.
  • Veggi PC, Cavalcanti RN, Meireles MAA. Production of phenolic-rich extracts from Brazilian plants using supercritical and subcritical fluid extraction: Experimental data and economic evaluation. J Food Eng. 2014;131:96-109.
  • Zoghbi MDGB, Oliveira J, Guilhon GMSP. The genus Mansoa (Bignoniaceae): A source of organosulfur compounds. Braz J Pharmacogn. 2009;19(3):795-804.

Publication Dates

  • Publication in this collection
    25 Nov 2022
  • Date of issue
    2022

History

  • Received
    09 July 2020
  • Accepted
    14 Mar 2021
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br