Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

One-step ultrasound-assisted recovery of yellow-orange-red natural coloring from defatted annatto seeds: A cleaner processing alternative.

The interest in natural colorants derived from sustainable processes has prompted research into obtaining bixin from defatted annatto (Bixa orellana L.) seeds. Bixin is a compound that imparts yellow-orange-red coloration, known for its high biodegradability, low toxicity, and wide industrial applicability. Meanwhile, high-intensity ultrasound (HIUS) technology has emerged as a promising method for extracting natural colorants, offering higher yields through shorter processes and minimizing thermal degradation. Although some studies have demonstrated the efficiency of HIUS technology in bixin extraction, research on the effects of acoustic cavitation on the properties of the colorant remains limited. Therefore, this study aimed to investigate the influence of HIUS-specific energy levels (0.02, 0.04, 0.12, and 0.20 kJ/g) on the chemical, physical, and morphological characteristics of annatto extracts containing bixin and geranylgeraniol. Single-step extractions of bixin using ethanol as a solvent were evaluated at various acoustic powers (4.6, 8.5, 14.5, and 20 W) and extraction times (0.5, 1, 3, and 5 min) to determine their impact on the yield of natural colorant extraction. Increasing the acoustic power from 4.6 to 20 W and extending the extraction time from 0.5 to 5 min resulted in higher yields of natural colorant, likely due to the effects of acoustic cavitation and increased heat under more intense conditions. However, elevated levels of mechanical and thermal energy did not affect the chemical properties of the colorant, as indicated by UV-Vis and FTIR spectra. Conversely, higher specific energies yielded colorants with a more intense red hue, consistent with increased bixin content, and altered the microstructure and physical state, as observed in X-ray diffractograms. Nevertheless, these alterations did not impact the solubility of the colorant. Therefore, employing a cleaner extraction procedure aided by one-step ultrasound facilitated the recovery of natural colorants and contributed to the biorefining of annatto seeds, enabling the production of a rich geranylgeraniol colorant through a sustainable approach.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

Effect of the Association of Fixed Oils from Abelmoschus esculentus (L.) Moench, Euterpe oleracea Martius, Bixa orellana Linné and Chronic SM® on Atherogenic Dyslipidemia in Wistar Rats.

Dyslipidemia presents high levels of serum cholesterol and is characterized as a risk factor for cardiovascular diseases, especially for the development of atherosclerosis. E. oleracea oil (OFEO), A. esculentus oil (OFAE), B. orellana oil (OFBO), and Chronic SM® granules (CHR) are rich in bioactive compounds with the potential to treat changes in lipid metabolism. This study investigated the effects of treatments with oils from A. esculentus, E. oleracea, B. orellana, and Chronic SM® on Cocos nucifera L. saturated-fat-induced dyslipidemia. The chromatographic profile showed the majority presence of unsaturated fatty acids in the tested oils. The quantification of tocotrienols and geranylgeraniol in OFBO and CHR was obtained. Treatments with OFEO, OFAE, OFBO, and CHR were able to significantly reduce glycemia, as well as hypertriglyceridemia, total cholesterol, and LDL-cholesterol, besides increasing HDL-cholesterol. The treatments inhibited the formation of atheromatous plaques in the vascular endothelium of the treated rats. The obtained results suggest that the OFEO, OFAE, OFBO, and CHR exhibit antidyslipidemic effects and antiatherogenic activity.

Investigation on the effect of antimicrobial photodynamic therapy as an adjunct for management of deep caries lesions-study protocol for a randomized, parallel groups, controlled clinical trial.

BACKGROUND: Alternatively to conventional treatments, chemo-mechanical caries removal agents can be used. A modality of treatment that has been increasing in dentistry is antimicrobial photodynamic therapy (aPDT). Bixa orellana is being researched for application in aPDT. This protocol aims to determine the effectiveness of aPDT with Bixa orellana extract in deep caries lesions. METHODS: A total of 160 teeth with deep occlusal dental caries will be selected and divided into 4 groups: G1 - control group (Caries removal with a low-speed drill); G2 - Partial Caries Removal with Papacarie™ (Fórmula e Ação, São Paulo, SP, Brazil); G3 - Partial Caries Removal with Papacarie™ and application Bixa orellana extract (20%) (Fórmula e Ação, São Paulo, SP, Brazil); G4 - Partial Caries Removal with Papacarie™ and application Bixa orellana extract (20%) with LED (Valo Cordless Ultradent®, South Jordan, UT, USA) (aPDT). After treatment, all the teeth will be restored with glass ionomer cement and followed up clinically and radiographically, with evaluations at immediately, 1 week, and 1, 3, 6, and 12 months. Dentin samples before and after treatment will be analyzed microbiologically. The efficacy of treatments will be assessed with microbiological (colony-forming units, before and after carious tissue removal), radiographic (integrity of the periapical area and eventual changes in the radiolucent zones), and clinical examinations (retention of the restorative material in the cavity and occurrence of secondary caries), as well as with the time required for the procedures and the need for anesthesia during the procedures. In case data distribution is normal, analysis of variance (ANOVA) will be used for both the dependent and independent variables. In case the data distribution is not normal, the Friedman test will be used for the dependent variables. For independent variables, the Kruskal-Wallis test will be used. DISCUSSION: Procedures using aPDT have been developed for the treatment of dental caries, but there are few controlled clinical trials in the literature confirming its efficacy. TRIAL REGISTRATION: This protocol is registered at ClinicalTrials.gov under the number NCT05236205 and it was first posted on 01/21/2022 and last updated on 05/10/2022.

Intramuscular compatibility of an injectable anti-inflammatory nanodispersion from a standardized Bixa orellana oil (Chronic®): a toxicological study in Wistar rats.

Bixa orellana L. is a plant popularly known as "ucurum", "annatto", and "achiote". It is native to South America, and its seeds are an abundant source of geranylgeraniol and tocotrienols. Nanoencapsulation is a valuable technique that can decrease the drug needed to achieve an effect, decreasing potential toxicity, side effects and potentiate the anti-inflammatory effect. This study aimed to evaluate the acute toxicity of an intramuscular application of a nanodispersion containing a standardized extract from the seeds of Bixa orellana (NBO) in Wistar rats. The chemical evaluation showed δ-tocotrienol at 0.725 ± 0.062 mg/mL (72.6 ± 0.9%). The stability study showed the nanoparticles had an average size from 53.15 ± 0.64 to 59.9 ± 3.63 nm, with a polydispersity index ranging from 0.574 ± 0.032 to 0.574 ± 0.32, Zeta potential from 18.26 ± 0.59 to 19.66 ± 1.45 mV. After testing the intramuscular application of NBO with doses from 1 to 5 mg/kg in animals, it was observed that the acute treatment did not elicit any toxic effects within this range. The dose of 10 mg/kg, although not affecting hematological and biochemical parameters (CPK, LDH, myoglobin, AST, ALT, TC, TG, glucose levels, creatinine, and urea), could induce some muscle tissue changes, including leukocyte infiltration, morphological chances, and potentially necrosis. In conclusion, the results showed that the treatments devoided toxicity between 1 and 5 mg/kg.

Dynamics of annatto pigment synthesis and accumulation in seeds of Bixa orellana L. revealed by integrated chemical, anatomical, and RNA-Seq analyses.

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3, and S4) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Consumption of Eggs Alone or Enriched with Annatto (Bixa orellana L.) Does Not Increase Cardiovascular Risk in Healthy Adults-A Randomized Clinical Trial, the Eggant Study.

Most atherosclerotic cardiovascular diseases can be prevented by modifying lifestyles, including unhealthy diets. Eggs contain important carotenoids that may impact cardiovascular risk. The lipid nature of eggs can improve the bioavailability of other carotenoids, such as Annatto (Bixa orellana L.), with reported antioxidant properties. Although numerous studies have shown that there is no association between egg consumption and cardiovascular risk, there is still controversy. In addition, there is limited information about Annatto's effects on human health. This study evaluated the association between egg consumption and its enrichment with Annatto in lipid biomarkers of cardiovascular disease. In a parallel clinical trial, one hundred and five (n = 105) men and women were randomized by age, sex, and body mass index (BMI), and distributed into three groups. Subjects consumed daily, for 8 weeks, either two eggs, two eggs with Annatto, or two egg whites. Plasma lipids were measured by enzymatic colorimetric methods, plasma apolipoproteins and lipoprotein subfractions and size by nuclear magnetic resonance. There were no differences between groups in age, sex, and BMI. No significant changes were found over time or between groups in plasma triglycerides, LDL cholesterol, HDL cholesterol, apolipoprotein (apo) A1, apo B, or lipoprotein subfraction concentrations. In healthy adults, the intake of two eggs a day, or two eggs with Annatto for eight weeks, did not generate adverse changes in cardiovascular risk markers.

Accumulation and decomposition of cultural residues of Theobroma grandiflorum, Paullinia cupana, Bixa orellana and forest in the southern region of Amazonas.

The litter deposited on the soil surface at various stages of decomposition is important for primary productivity that impacts the microbial communities and soil carbon storage. The objective of this study was to evaluate the accumulation and decomposition of cultural residues of Theobroma grandiflorum (Willd. ex. Spreng) Schum, Paullinia cupana (Mart.) Ducke, Bixa orellana L., and forest in the Amazon region. The study was carried out in the São Francisco settlement, Canutama in the south of Amazonas, in a randomized block experimental design, and the treatments consisted of four areas with different crops: 1 - P. cupana; 2 - T. grandiflorum; 3 - B. orellana; 4 - Native woodland area (forest), in time subdivided plots: 7, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, and 330 days after the distribution of the bags in the field, all with four repetitions. To evaluate the contribution and fractions of litter, conical collectors were used in each area, and collections were performed monthly in the period from March 2020 to February 2021. The estimate of the decomposition rate of the litter was done by quantifying the loss of mass, using litter bags, which allow for a direct analysis of the rate of decay over time. The forest and P. cupana environments presented the highest litter production, and greater deposition when compared to environments cultivated with T. grandiflorum and B. orellana. The forest and B. orellana areas showed the highest speed of decomposition, while the opposite situation occurred under T. grandiflorum and P. cupana cultivation.

Chemical composition and effects of ethanolic extract and gel of Cochlospermum regium (Schrank) Pilg. Leaves on inflammation, pain, and wounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Cochlospermum regium is well-known as "Algodãozinho do cerrado" in folk Brazilian medicine, and is used to fight infections, inflammation and skin disorders. AIM OF THE STUDY: To identify the phytochemical constituents and the effects of the ethanolic extract of C. regium leaves (EECR) on inflammation and pain, and the effects of C. regium gel (GEECR) on wound healing. MATERIALS AND METHODS: Animals were treated with EECR (30-300 mg/kg) or GEECR (1.25 and 2.5%) and studies were conducted using carrageenan-induced pleurisy and paw edema tests, formalin-induced pain model, and excision wound model. RESULTS: In total, 25 compounds, including quercitrin, methyl gallate, and 1,2,3,4,6-pentagalloylhexose, with highest detectability were identified. The treatments reduced leukocyte migration, nitric oxide production, protein extravasation, edema, mechanical hyperalgesia, pain in both phases (neurogenic and inflammatory), cold hypersensitivity, and improved wound closure and tissue regeneration. CONCLUSIONS: The present findings established the anti-inflammatory, anti-nociceptive, and wound healing potential of the leaves of C. regium, confirming the potential therapeutic effect of this plant.

Accumulation and decomposition of cultural residues of Theobroma grandiflorum, Paullinia cupana, Bixa orellana and forest in the southern region of Amazonas / Acúmulo e decomposição dos resíduos culturais de cupuaçu, guaraná, urucum e floresta na região sul do Amazonas

The litter deposited on the soil surface at various stages of decomposition is important for primary productivity that impacts the microbial communities and soil carbon storage. The objective of this study was to evaluate the accumulation and decomposition of cultural residues of Theobroma grandiflorum (Willd. ex. Spreng) Schum, Paullinia cupana (Mart.) Ducke, Bixa orellana L., and forest in the Amazon region. The study was carried out in the São Francisco settlement, Canutama in the south of Amazonas, in a randomized block experimental design, and the treatments consisted of four areas with different crops: 1 - P. cupana; 2 - T. grandiflorum; 3 - B. orellana; 4 - Native woodland area (forest), in time subdivided plots: 7, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, and 330 days after the distribution of the bags in the field, all with four repetitions. To evaluate the contribution and fractions of litter, conical collectors were used in each area, and collections were performed monthly in the period from March 2020 to February 2021. The estimate of the decomposition rate of the litter was done by quantifying the loss of mass, using litter bags, which allow for a direct analysis of the rate of decay over time. The forest and P. cupana environments presented the highest litter production, and greater deposition when compared to environments cultivated with T. grandiflorum and B. orellana. The forest and B. orellana areas showed the highest speed of decomposition, while the opposite situation occurred under T. grandiflorum and P. cupana cultivation.

A serrapilheira depositada na superfície do solo, em vários estágios de decomposição tem importância na produtividade primária que impacta nas comunidades microbianas e estocagem de carbono no solo. Objetivouse neste trabalho avaliar o acúmulo e decomposição dos resíduos culturais de cupuaçu, guaraná, urucum e floresta na região do Amazonas. O estudo foi realizado no assentamento São Francisco, Canutama no Sul do Amazonas, em delineamento experimental de blocos casualisados, sendo que os tratamentos constaram de quatro áreas com diferentes culturas: 1- Guaraná; 2 ­ Cupuaçu; 3 ­ Urucum; 4 ­ Área de mata nativa (floresta), em parcelas subdivididas no tempo: 07, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270 e 300, 330 dias após a distribuição das sacolas no campo, todas com 04 repetições. Para avaliar o aporte e frações dá serapilheira, foram utilizados coletores cônicos em cada área, sendo as coletas realizadas mensalmente no período de março de 2020 a fevereiro de 2021. A estimativa da taxa de decomposição da serrapilheira foi realizada pela quantificação da perda de massa, utilizando-se litter bags, os quais permitem analisar de forma direta a taxa de decaimento ao longo do tempo. Os ambientes de floresta e guaraná apresentaram as maiores produções de serapilheira, e maior deposição quando comparados aos ambientes cultivados com cupuaçu e urucum. As áreas de floresta e urucum apresentaram a maior velocidade de decomposição, já a situação inversa ocorreu sob o cultivo do cupuaçu e guaraná.

Yolk color and sensory analysis of eggs of common quails (Coturnix coturnix coturnix) fed sorghum-based diet with the addition of annatto seed meal (Bixa orellana L) / Coloração da gema e análise sensorial de ovos de codornas europeias (Coturnix coturnix coturnix) alimentadas com ração à base de sorgo com adição de farelo de urucum (Bixa orellana L)

This study has aimed to assess the effect of increasing levels of annatto (Bixa orellana L.) seed meal (AM) on yolk pigmentation and the sensory analysis of eggs of common quails fed sorghum-based diets when compared tocorn. Eighty female common quails (Coturnix coturnix coturnix) in the laying phase were used, being them aged from 251 to 316 days. There were three experimental periods of 21 days each (251-272; 273-294; 295-316 days). The quails were distributed in a completely randomized design with five treatments (T1 - corn-based feed; T2 - feed with 100% of sorghum instead of corn without the addition of AM; T3, T4, and T5 - feed with 100% of sorghum replacing corn with the addition of 0.5, 1.0, and 1.5% AM, respectively) and four repetitions. One hundred eggs from each treatment were used for analysis. Sensory evaluation was applied with an untrained panel with 20 evaluators. The eggs were boiled, peeled, and served. The panelists assessed the appearance, flavor, color, aroma, texture, and overall evaluation of the eggs. Annatto seed meal added to sorghum-based diets promoted linear increases in appearance, flavor, color, aroma, texture, and overall evaluation, evaluated during the sensory analysis (p=0.001). The addition of 1.5% AM in the sorghum-based diets of the common quails benefited the characteristics of appearance (4.50), flavor (4.50), color (4.55), aroma (4.25), and texture (4.55) of the assessed quail eggs, being considered more attractive and with greater acceptance (overall evaluation = 5.97) in relation to the other treatments tested. The inclusion of AM in the sorghum-based diets improved the yolk pigmentation of quail eggs in relation to the control treatments. The AM triggers positive effects on yolk pigmentation and sensory characteristics of common quail eggs.(AU)

Objetivou-se avaliar o efeito de níveis crescentes do farelo do resíduo da semente de urucum (Bixa orellana L.) (FU), sobre a pigmentação da gema e análise sensorial dos ovos de codornas europeias alimentadas com dietas à base de sorgo em substituição ao milho. Foram utilizadas 80 codornas europeias (Coturnix coturnix coturnix) fêmeas, na fase de postura, no período de 251 a 316 dias de idade. Foram três períodos experimentais de 21 dias cada (251-272; 273-294; 295-316 dias). As codornas foram distribuídas em delineamento inteiramente casualizado, com cinco tratamentos (T1 - ração à base de milho; T2 - ração com 100% de sorgo em substituição ao milho sem a adição de FU; T3, T4 e T5 ­ ração com 100% de sorgo em substituição ao milho com a adição de 0,5; 1,0 e 1,5% de FU) e quatro repetições. Cem ovos de cada tratamento foram utilizados para a análise. A avaliação sensorial foi aplicada em painel não treinado de 20 avaliadores. Os ovos foram cozidos, descascados e servidos. A aparência, sabor, cor, odor, textura e avaliação global, foram avaliadas pelos painelistas. A farinha de semente de urucum adicionada à ração à base de sorgo promoveu aumentos lineares nos atributos aparência, sabor, cor, odor, textura e avaliação global, avaliados durante a análise sensorial (p=0,001). A adição de 1,5% de FU em dietas a base de sorgo beneficiou as características de aparência (4,50), sabor (4,50) cor (4,55), aroma (4,25) e textura (4,55) dos ovos de codorna avaliados, sendo considerados mais atrativos e com maior aceitação (avaliação global = 5,97), em relação aos demais tratamentos testados. A inclusão do FU nas rações à base de sorgo melhorou a pigmentação da gema dos ovos de codorna em relação aos tratamentos controle. O AM desencadeia efeitos positivos na pigmentação da gema e nas características sensoriais de ovos de codornas comuns.(AU)

Green Extraction of Annatto Seed Oily Extract and Its Use as a Pharmaceutical Material for the Production of Lipid Nanoparticles.

This work developd nanomaterials formulated from annatto seed oily extract (ASE), myristic acid (tetradecanoic acid), and their fatty acid esters. The annatto seed oily extract was obtained using only soybean oil (ASE + SO) and Brazil nut oil (ASE + BNO). The UV/VIS analysis of the oily extracts showed three characteristic peaks of the bixin molecule at 430, 456 and 486 nm. The lipid nanoparticles obtained using myristic acid and ASE + BNO or only BNO showed better results than the oil soybean extract, i.e., the particle size was <200 nm, PDI value was in the range of 0.2−0.3, and had no visual physical instability as they kept stable for 28 days at 4 °C. Lipid nanoemulsions were also produced with esters of myristic acid and ASE + BNO. These fatty acid esters significantly influenced the particle size of nanoemulsions. For instance, methyl tetradecanoate led to the smallest particle size nanoemulsions (124 nm), homogeneous size distribution, and high physical stability under 4 and 32 °C for 28 days. This work demonstrates that the chemical composition of vegetable oils and myristic acid esters, the storage temperature, the chain length of fatty acid esters (FAE), and their use as co-lipids improve the physical stability of lipid nanoemulsions and nanoparticles from annatto seed oily extract.

Organic Electronics from Nature: Computational Investigation of the Electronic and Optical Properties of the Isomers of Bixin and Norbixin Present in the Achiote Seeds.

Organic compounds have been employed in developing new green energy solutions with good cost-efficiency compromise, such as photovoltaics. The light-harvesting process in these applications is a crucial feature that still needs improvements. Here, we studied natural dyes to propose an alternative for enhancing the light-harvesting capability of photovoltaics. We performed density functional theory calculations to investigate the electronic and optical properties of the four natural dyes found in achiote seeds (Bixa orellana L.). Different DFT functionals, and basis sets, were used to calculate the electronic and optical properties of the bixin, norbixin, and their trans-isomers (molecules present in Bixa orellana L.). We observed that the planarity of the molecules and their similar extension for the conjugation pathways provide substantially delocalized wavefunctions of the frontier orbitals and similar values for their energies. Our findings also revealed a strong absorption peak in the blue region and an absorption band over the visible spectrum. These results indicate that Bixa orellana L. molecules can be good candidates for improving light-harvesting in photovoltaics.

Potential of the Compounds from Bixa orellana Purified Annatto Oil and Its Granules (Chronic®) against Dyslipidemia and Inflammatory Diseases: In Silico Studies with Geranylgeraniol and Tocotrienols.

Some significant compounds present in annatto are geranylgeraniol and tocotrienols. These compounds have beneficial effects against hyperlipidemia and chronic diseases, where oxidative stress and inflammation are present, but the exact mechanism of action of such activities is still a subject of research. This study aimed to evaluate possible mechanisms of action that could be underlying the activities of these molecules. For this, in silico approaches such as ligand topology (PASS and SEA servers) and molecular docking with the software GOLD were used. Additionally, we screened some pharmacokinetic and toxicological parameters using the servers PreADMET, SwissADME, and ProTox-II. The results corroborate the antidyslipidemia and anti-inflammatory activities of geranylgeraniol and tocotrienols. Notably, some new mechanisms of action were predicted to be potentially underlying the activities of these compounds, including inhibition of squalene monooxygenase, lanosterol synthase, and phospholipase A2. These results give new insight into new mechanisms of action involved in these molecules from annatto and Chronic®.

Bixa orellana L. (Achiote, Annatto) as an antimicrobial agent: A scoping review of its efficiency and technological prospecting.

ETHNOPHARMACOLOGICAL RELEVANCE: Bixa orellana L. is reported to have numerous applications in traditional medicine and pharmacological properties such as wound healing, analgesic, hemostatic, and antioxidant activities. Recently, the literature has shown scientific interest of its antimicrobial properties aiming the development of cost-effective phytotherapeutic agents. However, no literature are available in witch the antimicrobial and technological prospecting are summarized. AIM OF STUDY: This study aimed to systematically review articles and patents related to the antimicrobial activity of B. orellana. METHODS: The review followed the guidelines proposed by The Joanna Briggs Institute and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Two reviewers performed a literature search up to November 2021 in eight databases: Medline (PubMed), Scopus, Scifinder, Web of Science, Cochrane, Embase, Scielo, and Biblioteca Virtual em Saúde. The following databases for the patent search were analyzed: United States Patent and Trademark Office (USPTO), Google Patents, National Institute of Industrial Property (INPI), World Intellectual Property Organization (WIPO), and Espacenet (European Patent Office, EPO). The grey literature was searched using the ProQuest Dissertations and Periódicos Capes Theses database. The methodological quality and risk of bias in the included studies were carried out using Review Manager (RevMan) 5.3.5. RESULTS: After analyzing the 47 studies and five patents fulfilled all the criteria and were included in the present investigation. The evidence suggests that this herbal medicine is effective against several fungi, Gram-positive and Gram-negative bacteria, being more effective to Gram-negative bacteria. Regarding the risk of bias and methodological quality analysis, most studies a high risk of bias related to random sequence generation and allocation concealment. CONCLUSION: Up to now, the evidence in the literature suggests that the use of B. orellana preparations for antimicrobial preparations has some effectiveness. However, further research is needed using standard B. orellana preparations to determine their effectiveness as antimicrobial agents and expanding their application at an industrial level, in addition in vivo studies are needed for this confirmation.

In vitro antimicrobial activity of Bixa orellana L . Leaves extract against anaerobic bacteria associated to bacterial vaginosis and Lactobacillus spp. / Actividad antimicrobiana in vitro del extracto de hojas de Bixa orellana L. contra bacterias anaerobias asociadas a la vaginosis bacteriana y Lactobacillus spp.

OBJECTIVE.: Motivation for the study: bacterial vaginosis is a bacterial infection that frequently affects women of reproductive age. The treatment is based on synthetic antimicrobials. Bixa orellana L. possesses antimicrobial properties and could represent a potential non-synthetic therapeutic alternative. Main findings: in vitro results suggest that, methanolic extract of Bixa orellana L. leaves possesses potential antimicrobial properties against bacteria associated to bacterial vaginosis. Implications: to identify new sources with therapeutic potential, and to promote research, discovery, and characterization of non-synthetic antimicrobials. To describe the in vitro antimicrobial activity of the methanolic extract of Bixa orellana L. leaves against anaerobic bacteria associated to bacterial vaginosis and Lactobacillus spp. MATERIALS AND METHODS.: Eight ATCC reference strains; Gardnerella vaginalis, Prevotella bivia, Peptococcus niger, Peptostreptococcus anaerobius, Mobiluncus curtisii, Atopobium vaginae, Veillonella parvula, and Lactobacillus crispatus, and twenty-two clinical isolates; eleven Gardnerella vaginalis and eleven Lactobacillus strains, were included in the study. The antimicrobial susceptibility was determined by the agar diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by using agar dilution and a modified dilution plating method, respectively. RESULTS.: All ATCC reference strains showed high levels of susceptibility to the extract, except P. vibia, V. parvula and L. crispatus. Interestingly, all G. vaginalis clinical isolates and the G. vaginalis ATTC strain were the most susceptible to the extract, given their low MIC (1.0 - 2.0 mg/mL) and MBC (1.0 - 4.0 mg/mL) values, whereas, the Lactobacillus spp. clinical isolates and the L. crispatus ATCC strain were the least susceptible bacteria given their high MIC (32.0 mg/mL) and MBC (≥ 32.0 mg/mL) values. CONCLUSIONS.: In vitro experiments suggest that the extract possesses selective antimicrobial properties given its high activity against bacterial vaginosis-associated anaerobic bacteria and low activity against Lactobacillus species.

OBJETIVOS: Motivación para realizar el estudio: la vaginosis bacteriana es una infección bacteriana que afecta de forma frecuente a las mujeres en edad reproductiva. El tratamiento se basa en antimicrobianos sintéticos. Bixa orellana L. posee propiedades antimicrobianas y podría representar una potencial alternativa terapéutica no sintética. Principales hallazgos: los resultados in vitro sugieren que el extracto metanólico de las hojas de Bixa orellana L. posee potenciales propiedades antimicrobianas contra las bacterias asociadas a vaginosis bacteriana. Implicancias: identificar nuevas fuentes con potencial terapéutico, y promover la investigación, descubrimiento y caracterización de antimicrobianos no sintéticos. Describir la actividad antimicrobiana in vitro del extracto metanólico de las hojas de Bixa orellana L. contra las bacterias anaerobias asociadas a la vaginosis bacteriana y Lactobacillus spp. MATERIALES Y MÉTODOS: . Se incluyeron en el estudio ocho cepas de referencia ATCC; Gardnerella vaginalis, Prevotella bivia, Peptococcus niger, Peptostreptococcus anaerobius, Mobiluncus curtisii, Atopobium vaginae, Veillonella parvula y Lactobacillus crispatus, y 22 aislamientos clínicos; once aislados de Gardnerella vaginalis y once aislados de Lactobacillus. La susceptibilidad antimicrobiana se determinó mediante el método de difusión en agar. La concentración mínima inhibitoria (CMI) y la concentración bactericida mínima (CBM) fueron determinadas utilizando el método de dilución en agar y un método de dilución modificado, respectivamente. RESULTADOS: . Todas las cepas de referencia ATCC tuvieron un alto nivel de susceptibilidad al extracto, con excepción de P. vibia, V. parvula y L. crispatus. Interesantemente, los aislamientos clínicos de G. vaginalis y la cepa ATCC de G. vaginalis fueron los más susceptibles al extracto dados los bajos valores de CMI (1,0 - 2,0 mg/mL) y CBM (1,0 - 4,0 mg/mL), mientras que, los aislamientos clínicos de Lactobacillus spp. y la cepa ATCC de L. crispatus fueron los menos susceptibles debido a los altos valores de CMI (32,0 mg/mL) y CBM (≥ 32,0 mg/mL). CONCLUSIONES: . Los experimentos in vitro sugieren que el extracto posee propiedades antibacterianas selectivas dada su alta actividad contra bacterias anaerobias asociadas a vaginosis bacteriana y baja actividad contra especies de Lactobacillus.

Germination potential and methods for overcoming seed dormancy for domesticated and wild annatto populations after two years of storage / Potencial de germinação e métodos de superação da dormência de sementes para populações domesticadas e selvagens de urucum após dois anos de armazenamento

Annatto (Bixa orellana L.) is an arboreal species domesticated in Amazonia from its wild ancestor (B. orellana var. urucurana). Bixin extracted from its orthodox seeds is a natural dye widely used in the food industry. This study evaluated methods to overcome seed dormancy and determine the germination potential, comparing domesticated and wild annatto populations. Seeds from two domesticated-type populations and two families of a wild-type population, stored for two years after field collection, were submitted to five treatments to overcome dormancy: T1 - control; T2 - mechanical scarification (with sandpaper); T3 - mechanical scarification (with sandpaper) + immersion in water at 36 ºC overnight (12 hours); T4 - immersion in water at room temperature (23 ºC, on average) for 24 h; T5 - immersion in concentrated sulfuric acid (95 - 98%) for 15 min + running water for 3 min. Highly significant differences (P < 0.001) were observed in the germination percentage of annatto seeds between wild and domesticated types, and among the treatments tested. Domesticated types showed higher germination percentage (10 - 58%) over all treatments when compared to the wild type (0 - 44%). The best treatments were those performed with mechanical scarification. Given the simplicity, we concluded that mechanical scarification with sandpaper is a good alternative to overcome dormancy of annatto seeds.

O urucum (Bixa orellana L.) é uma espécie arbórea domesticada na Amazônia a partir de seu ancestral selvagem (B. orellana var. urucurana). Das suas sementes ortodoxas, é extraído a bixina, corante bastante utilizado na alimentação. Este estudo teve como objetivos avaliar métodos de superação da dormência e determinar o potencial de germinação de sementes, comparando populações domesticadas e selvagens. Sementes de duas populações domesticadas e duas famílias de uma população selvagem, armazenadas por dois anos após a coleta, foram submetidas a cinco tratamentos de superação da dormência: T1 - controle; T2 - escarificação mecânica (com lixa); T3 - escarificação mecânica (com lixa) + imersão em água a 36 ºC durante a noite (12 horas); T4 - imersão em água à temperatura ambiente (23 ºC, em média) por 24 h; T5 - imersão em ácido sulfúrico concentrado (95 - 98%) por 15 min + água corrente por 3 min. Foram observadas diferenças altamente significativas (P < 0,001) no potencial de germinação de sementes entre domesticadas e selvagens, e entre os tratamentos testados. As populações domesticadas apresentaram maior porcentagem de germinação (10 - 58%) em comparação com a de urucum selvagem (0 - 44%). Os melhores tratamentos foram os realizados com escarificação mecânica. Considerando sua simplicidade, concluímos que a escarificação mecânica com lixa representa boa alternativa para superar a dormência do urucum.

Co-Treatment of Purified Annatto Oil (Bixa orellana L.) and Its Granules (Chronic®) Improves the Blood Lipid Profile and Bone Protective Effects of Testosterone in the Orchiectomy-Induced Osteoporosis in Wistar Rats.

This study aimed to evaluate and compare the effects of co-treatment with purified annatto oil (PAO) or its granules (GRA, Chronic®) with that of testosterone on the orchiectomy-induced osteoporosis in Wistar rats. After surgery, rats were treated from day 7 until day 45 with testosterone only (TES, 7 mg/kg, IM) or TES + PAO or GRA (200 mg/kg, p.o.). The following parameters were evaluated: food/water intake, weight, HDL, LDL, glucose, triglycerides (TG), total cholesterol (TC), alkaline phosphatase levels, blood phosphorus and calcium contents, femur weight, structure (through scanning electron microscopy), and calcium content (through atomic absorption spectrophotometry). Our results show that orchiectomy could significantly change the blood lipid profile and decrease bone integrity parameters. Testosterone reposition alone could improve some endpoints, including LDL, TC, bone weight, and bone calcium concentration. However, other parameters were not significantly improved. Co-treatment with PAO or GRA improved the blood lipid profile and bone integrity more significantly and improved some endpoints not affected by testosterone reposition alone (such as TG levels and trabeculae sizes). The results suggest that co-treatment with annatto products improved the blood lipid profile and the anti-osteoporosis effects of testosterone. Overall, GRA had better results than PAO.

Antimicrobial and physical properties of experimental endodontic sealers containing vegetable extracts.

To assess the antimicrobial activity and the physical properties of resin-based experimental endodontic sealers with the incorporation of vegetable extracts obtained from Bixa orellana, Mentha piperita, and Tagetes minuta species. The extracts were obtained and characterized by gas chromatography-mass spectrometry (GC-MS), and minimum inhibitory concentration (MIC) against Streptococcus mutans, Enterococcus faecalis, and Candida albicans. The extracts were individually incorporated into a dual-cure experimental sealer at a mass concentration of 0.5%. A commercial reference RealSeal was used. The sealers were evaluated by measuring the setting time, degree of conversion, dimensional stability, radiopacity, flow, and film thickness of these materials, also and its antimicrobial effect was evaluated using the direct contact test. Data were statistically analyzed by analysis of variance and Tukey's post-hoc test at α = 0.05 significance level. The physical properties were not influenced by the addition of the vegetable extracts (p > 0.05). For S. mutans, only T. minuta and B. orellana groups presented antibacterial activity after 24 h of contact (p < 0.05). All extracts evidenced an antibacterial effect against E. faecalis (p < 0.05). The experimental sealers hold promise as a novel vegetable sealer with great antimicrobial activity and also great physical-mechanical properties. Nonetheless, more studies are needed.