Evidence for the efficacy of anti-inflammatory plants used in Brazilian traditional medicine with ethnopharmacological relevance.

ETHNOPHARMACOLOGICAL RELEVANCE: When exacerbated, inflammatory processes can culminate in physical and emotional disorders and, if not stopped, can be lethal. The high prevalence of inflammation has become a public health problem, and the need for new drugs to treat this pathology is imminent. The use of medicinal plants has emerged as an alternative, and a survey of data that corroborates its application in inflammatory diseases is the starting point. Furthermore, Brazil harbors a megadiversity, and the traditional use of plants is relevant and needs to be preserved and carefully explored for the discovery of new medicines. AIM OF THE STUDY: This review sought to survey the medicinal plants traditionally used in Brazil for the treatment of inflammatory processes and to perform, in an integrative way, a data survey of these species and analysis of their phytochemical, pharmacological, and molecular approaches. MATERIALS AND METHODS: Brazilian plants that are traditionally used for inflammation (ophthalmia, throat inflammation, orchitis, urinary tract inflammation, ear inflammation, and inflammation in general) are listed in the DATAPLAMT database. This database contains information on approximately 3400 native plants used by Brazilians, which were registered in specific documents produced until 1950. These inflammatory disorders were searched in scientific databases (PubMed/Medline, Scopus, Web of Science, Lilacs, Scielo, Virtual Health Library), with standardization of DECS/MESH descriptors for inflammation in English, Spanish, French, and Portuguese, without chronological limitations. For the inclusion criteria, all articles had to be of the evaluated plant species, without association of synthesized substances, and full articles free available in any of the four languages searched. Duplicated articles and those that were not freely available were excluded. RESULTS: A total of 126 species were identified, culminating in 6181 articles in the search. After evaluation of the inclusion criteria, 172 articles representing 40 different species and 38 families were included in the study. Comparison of reproducibility in intra-species results became difficult because of the large number of extraction solvents tested and the wide diversity of evaluation models used. Although the number of in vitro and in vivo evaluations was high, only one clinical study was found (Abrus precatorius). In the phytochemical analyses, more than 225 compounds, mostly phenolic compounds, were identified. CONCLUSION: This review allowed the grouping of preclinical and clinical studies of several Brazilian species traditionally used for the treatment of many types of inflammation, corroborating new searches for their pharmacological properties as a way to aid public health. Furthermore, the large number of plants that have not yet been studied has encouraged new research to revive traditional knowledge.

Author Correction: Amaryllidaceae alkaloids: identification and partial characterization of montanine production in Rhodophiala bifida plant.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Amaryllidaceae alkaloids: identification and partial characterization of montanine production in Rhodophiala bifida plant.

Rhodophiala bifida (R. bifida) is a representative of the Amaryllidaceae plant family and is rich in montanine, an alkaloid with high pharmaceutical potential. Despite the interest in these compounds, many steps of the biosynthetic pathway have not been elucidated. In this study, we identified the alkaloids produced in different organs of R. bifida under different growth conditions, set up the conditions for in vitro R. bifida regeneration and initiated the molecular characterization of two R. bifida genes involved in alkaloids biosynthesis: the Norbelladine 4'-O-Methyltransferase (RbN4OMT) and the Cytochrome P450 (RbCYP96T). We show that montanine is the main alkaloid produced in the different R. bifida organs and developed a direct organogenesis regeneration protocol, using twin-scale explants cultivated on media enriched with naphthalene acetic acid and benzyladenine. Finally, we analyzed the RbN4OMT and RbCYP96T gene expressions in different organs and culture conditions and compared them to alkaloid production. In different organs of R. bifida young, adult and regenerated plants, as well as under various growing conditions, the transcripts accumulation was correlated with the production of alkaloids. This work provides new tools to improve the production of this important pharmaceutical compound and for future biotechnological studies.

Trifolium pratense: Friable calli, cell culture protocol and isoflavones content in wild plants, in vitro and cell cultures analyzed by UPLC

Abstract Trifolium pratense L., Fabaceae, is a rich source of isoflavones and has become the focus of several studies related to its phytoestrogenic activity. The aim of this study was to establish germination and cell cultures protocol for T. pratense and quantify isoflavones content in cell cultures, in vitro cultured and wild plants harvested in two different seasons. Murashige Skoog medium supplemented with naphthalene acetic acid and kinetin was able to produce the highest formation of friable calli. Calli cultures were analyzed qualitatively after 60 days of culture, and in vitro plants after 30, 45 and 60 days of cultivation. The chemical analysis was performed by ultra performance liquid chromatography, using the linearity curves of daidzein, genistein, formononetin and biochanin A as standards. The concentrations of isoflavones detected in wild plants were different in the two harvest periods and contrasted in content when compared to the in vitro plants. Cell cultures exhibited diverse profiles and concentration of isoflavones, none of which presented the isoflavonoid biochanin A. Pectinase was used to promote reduction of clumps and ended up altering the characteristics of secondary metabolites production in some cultures. Formononetin showed higher concentration in wild red clover samples (15.407 mg g-1), and in the in vitro grown plants the highest concentration was daidzein (17.591 mg g-1) at 60 days. The methods used for this research were effective, and the red clover plants of the analyzed variety can be cultivated in vitro aiming the commercial productivity by having contents greater than or equal to the wild plants in the periods studied, even without the use of elicitors during the cultivation.

Hydroxypropyl-ß-cyclodextrin-containing hydrogel enhances skin formononetin permeation/retention.

OBJECTIVES: This study was aimed to investigate the in vitro permeation potential of hydrogel formulations containing the isoflavones formononetin and biochanin A and cyclodextrins in different combinations. METHODS: The permeation assay was performed using porcine skin discs on Franz diffusion cells model. The isoflavone contents of the formulations were quantified in the different layers of the skin using a validated HPLC-PDA method. KEY FINDINGS: The isoflavones individually incorporated into the formulations showed high permeation potential, especially formononetin, after the incorporation of hydroxypropyl-ß-cyclodextrin that enhanced its permeation in the epidermis and dermis. Biochanin A showed 2.7 times of permeation capacity in the epidermis and dermis mainly after incorporation of cyclodextrins in the formulations. Formononetin showed reduction in its permeation when incorporated in the formulations together to biochanin A, showing the absence of synergism. CONCLUSIONS: Our results indicated a noticeable skin permeation promoting effect of HPßCD in formononetin formulation. Furthermore, formononetin and biochanin A can permeate the skin being mostly retained in the epidermis and dermis, revealing its potential use in cosmetic preparations intended to prevent skin aging.

Mevalonolactone: an inhibitor of Staphylococcus epidermidis adherence and biofilm formation.

Staphylococcus epidermidis, a commensal microorganism at the human skin and mucosae, is nowadays considered an important opportunistic pathogen related to nosocomial infections on indwelling medical devices due biofilm formation. Bacterial biofilms are the worst aspect in the treatment of infections and now efforts have been made in the search for new molecular entities to overcome this situation. In this work, a compound isolated from marine associated fungi was capable to interfere with the adherence and biofilm formation of S. epidermidis. This compound, identified as mevalonolactone, showed significant inhibition of S. epidermidis ATCC 35984 biofilm formation, without antibacterial activity, evaluated by crystal violet assay, turbidimetric assay and scanning electron microscopy. When assayed against 12 clinical isolates of S. epidermidis, this compound exhibited both biofilm inhibition and antimicrobial activity, but no activity against gram-negative bacteria was observed. Therefore, when this constitutive molecule is added in the antibiofilm and antibacterial assays, it might act as an important agent against this pathogen, contributing to the arsenal of antibiofilm compounds.

Dipeptide cis-cyclo(Leucyl-Tyrosyl) produced by sponge associated Penicillium sp. F37 inhibits biofilm formation of the pathogenic Staphylococcus epidermidis.

Infections associated to microbial biofilms are involved in 80% of human infections and became a challenge concerning public health. Infections related to Staphylococcus epidermidis biofilms are presently commonly associated to medical devices, increasing treatment costs for this type of infection. Alternatives to eliminate this kind of disease have been employed in screening programs using diverse marine-derived fungi source of bioactive compounds capable to combat biofilm formation. In this work was isolated the dipeptide cis-cyclo(Leucyl-Tyrosyl) from a sponge associated Penicillium sp. possessing a remarkable inhibition up to 85% of biofilm formation without interfering with bacterial growth, confirmed by scanning electron microscopy. This is the first demonstration that cis-cyclo(Leucyl-Tyrosyl) is able to specifically inhibit biofilm formation adding another aspect to the broad spectrum of bioactivities of cyclic dipeptides.

Anti-Trichomonas vaginalis activity of marine-associated fungi from the South Brazilian Coast.

Trichomonas vaginalis is the causative agent of trichomonosis, the most common non-viral sexually transmitted disease. Infection with this protozoan may have serious consequences, especially for women. Currently, 5-nitroimidazole drugs are the treatment of choice for trichomonosis, but the emergence of resistance has limited the effectiveness of this therapy. In this context, this study aimed to evaluate the anti-T. vaginalis activity of marine-associated fungi found in the South Brazilian Coast. A total of 42 marine-associated fungal species (126 filtrate samples) isolated from 39 different marine organisms, mainly sponges, were selected to be screened against T. vaginalis. Of these, two filtrate samples from Hypocrea lixii F02 and Penicillium citrinum F40 showed significant growth-inhibitory activity (up to 100%) against ATCC 30236 and fresh clinical isolates, including a metronidazole-resistant isolate. Minimum inhibitory concentration (MIC) values of H. lixii F02 and P. citrinum F40 samples for all isolates tested, including the metronidazole-resistant isolate, were 2.5 mg/mL. The kinetic growth curve showed that the filtrate samples were able to reduce the density of parasites to zero within 24 h of incubation, which was confirmed by microscopy. Both fungal filtrate samples exhibited no hemolytic activity, and the P. citrinum F40 filtrate sample showed low cytotoxicity against Vero cells. These data suggest that marine-associated fungi from the South Brazilian Coast may produce potential candidates for further investigation and possible use in the treatment of metronidazole-resistant trichomonosis.

Comparative analysis of Sambucus nigra and Sambucus australis flowers: development and validation of an HPLC method for raw material quantification and preliminary stability study.

This work was designed to develop a simple, effective, and reliable LC system to identify a chemical marker and compare Sambucus nigra L. and Sambucus australis Cham. et Schltdl. flower extracts (American and European elder). Rutin was the main constituent of both species. The developed method showed a linear response in the range of 10 to 45 microg x mL(-1) for rutin and 1.75 to 3.25 microg x mL(-1) for samples of the Sambucus species. Precision was determined and the relative standard deviations were 1.75 % for HSN and 1.28 % for HSA for intraday precision and 1.28 % and 1.51 % for inter-day precision, respectively, while accuracy was 97.9 % for HSN and 99.41 % for HSA. Quantification and detection limits as well as robustness were determined, presenting adequate results. The LC method showed an adequate performance for the separation of flavonoid glycosides in S. nigra and S. australis extracts, since the presence of interference had been previously evaluated. The analysis of thirty different samples of S. NIGRA and S. australis of different origins did not show significant variability among them. An accelerated stability study revealed a significant decrease in the first 30 days reaching 57 % in 90 days for S. australis samples and a total decrease of 25 % in 90 days for S. nigra samples, considering rutin as the chemical marker. These results will contribute to quality control analysis routines of these raw materials in pharmaceutical production facilities.

Caracterização farmacobotânica das espécies de Sambucus (Caprifoliaceae) utilizadas como medicinais no Brasil: Parte II. Sambucus australis Cham. & Schltdl. / Pharmacobotany of Sambucus species (Caprifoliaceae), used in traditional medicine in Brazil: Part II. Sambucus australis Cham. & Schltdl

Sambucus australis Cham. & Schltdl. (sabugueiro-do-rio-grande, Caprifoliaceae) é uma planta medicinal descrita apenas na primeira edição da Farmacopéia Brasileira. A espécie é nativa no Brasil, e suas flores são utilizadas na medicina popular, na forma de infusão ou decocção, como diuréticas, antipiréticas, antiinflamatórias, laxativo leve e no tratamento de doenças do aparelho respiratório. Visando elaborar uma monografia farmacopéica atualizada e comparativa com a de Sambucus nigra L., de origem européia, foram estabelecidos os caracteres botânicos macro e microscópicos, através da metodologia clássica utilizada em morfoanatomia vegetal. São características macroscópicas: flores morfologicamente monoclinas; corola de sete a dez milímetros de diâmetro; pétalas com cinco, raro quatro nervuras paralelas; estames curtos e longos; gineceu em regra com cinco lóculos. São características microscópicas: cutícula espessa e estriada; ausência de idioblastos de areia cristalina de oxalato de cálcio; estômatos anomocíticos; brácteas anfiestomáticas; sépalas hipoestomáticas; pétalas anfi-hipoestomáticas; células epidérmicas da face abaxial de sépalas e pétalas retilíneas a sinuosas; células epidérmicas do filete alongadas e de paredes retilíneas; tricomas tectores e glandulares de diferentes tipos; brácteas, sépalas e pétalas com mesofilo homogêneo; sistema vascular representado por feixes colaterais ou agrupamentos de elementos xilemáticos; presença de gotas lipídicas em todas as peças.

Sambucus australis Cham. & Schltdl. (sabugueiro-do-rio-grande, Caprifoliaceae) is a medicinal plant described only in the first edition of Brazilian Pharmacopoeia. This species is native to Brazil, and its flowers are used in the folk medicine, with diuretic purpose, antipiretic, anti-inflammatory, mild laxative and to treat respiratory diseases, on infusions or decoctions forms. Aiming at elaborating an updated pharmacopoeical monograph and comparing it with the monograph of Sambucus nigra L., from Europe, it was established the macro and microscopic botanical characters, through the classic methodology used in plant morpho-anatomy. The macroscopic characters are: morphologic monoclin flowers; corolla with seven to ten millimeters of diameter; petals with five, rarely four parallel veins; shorter and longer stamens; ovary oftenly with five locules. The main microscopic characters are: thickned and striated cuticule; absense of idioblasts with calcium oxalate sandy cristals; anomocytic stomatas; amphystomatic bracts; hypostomatic sepals; amphi-hypostomatic petals; wavy and straight epidermal cells in the lower surface of sepals and petals; filaments epidermal cells with narrow and straight walls; glandular and non-glandular trichomes of different types; homogeneous mesophyll in bracts, sepals and petals; vascular system represented by both collateral bundles and xylematic elements; volatile and non volatile oil globules in all structures.

Caracterização farmacobotânica das espécies de Sambucus (Caprifoliaceae) utilizadas como medicinais no Brasil. Parte I. Sambucus nigra L. / Pharmacobotany characterization of Sambucus species (Caprifoliaceae), used in traditional medicine in Brazil. Part I. Sambucus nigra L

O sabugueiro (Sambucus nigra L., Caprifoliaceae) é uma planta medicinal descrita em farmacopéias estrangeiras, não constando da Farmacopéia Brasileira. A espécie é de origem européia e suas flores são comercializadas in natura sob o nome farmacopéico Sambuci flos, para uso na forma de infusão ou decocção, como diuréticas, antipiréticas, antiinflamatórias, laxativo leve e no tratamento de doenças do aparelho respiratório. Visando elaborar uma monografia farmacopéica atualizada, foram estabelecidos os caracteres botânicos macro e microscópicos, através da metodologia clássica utilizada em morfoanatomia vegetal. São características macroscópicas: flores monoclinas, iguais; corola de três a cinco milímetros de diâmetro; pétalas com três a quatro nervuras paralelas; estames iguais; gineceu em regra com três lóculos. São características microscópicas: cutícula espessa e estriada; presença de idioblastos de areia cristalina de oxalato de cálcio em brácteas, sépalas e pétalas; estômatos anomocíticos; brácteas hipoestomáticas; sépalas anfiestomáticas; pétalas anfi-hipoestomáticas; células epidérmicas da face abaxial de sépalas e pétalas muito sinuosas; células epidérmicas do filete alongadas e de paredes retilíneas; tricomas tectores e glandulares de diferentes tipos em todas as peças; brácteas, sépalas e pétalas com mesofilo homogêneo; sistema vascular representado por feixes colaterais ou agrupamentos de elementos xilemáticos; presença de gotas lipídicas em todas as peças.

The Elder tree (Sambucus nigra L., Caprifoliaceae) is a medicinal plant described in international pharmacopoeias, not yet mentioned in the Brazilian Pharmacopoeia. The flowers of this European species are commercialized in natura by the pharmacopeial name of Sambuci flos, with diuretic, antipyretic, anti-inflammatory and mild laxative purposes, and to treat respiratory diseases, through infusions or decoctions forms. Aiming at elaborating an updated pharmacopeial monograph, the macro and microscopic botanic characters were established, with the classic methodology used in plant morpho-anatomy. The macroscopic characters are: monoclin and equal flowers; corolla with three to five millimeters of diameter; petals with three to four parallel veins; equal stamens; ovary often with three locules. The main microscopic characters are: thickened and striated cuticle; anomocytic stomatas; bracts, sepals and petals with sandy crystals of calcium oxalate idioblasts; hypostomatic bracts; amphystomatic sepals; amphi-hypostomatic petals; strongly wavy epidermal cells in the lower surface of sepals and petals; filaments epidermal cells with narrow and straight walls; glandular and non-glandular trichomes of different types in all pieces; homogeneous mesophyll in bracts, sepals and petals; vascular system represented by both collateral bundles and xylematic elements; volatile and non-volatile oil globules in all structures.