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BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

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Many biological activities have been reported for the Ilex genus. However, few studies in the literature have reported on guayusa. To address this gap in our knowledge, chemical analysis of guayusa leaves was made. Extracts were obtained by applying Soxhlet, maceration, supercritical CO2 and pressurised liquid extraction techniques, using water and ethanol as solvent/cosolvent. Extracts were evaluated for their phenolic content and antioxidant capacity. The chemical profile was obtained from HPLC. In raw guayusa leaves were identified caffeine (2.27 ± 0.05%), protein (15.31 ± 0.07%) and lipids (11.81 ± 0.14%). Extracts presented the highest phenolic content (156.56 ± 1.32 mg GAE g-1) and the best antioxidant activity (EC50= 61.85 ± 0.21 µg mL-1) when water was used as solvent/cosolvent. Through HPLC, three main substances were determined and quantified in the extracts: caffeine, theobromine and 5-caffeoylquinic acid. Based on these results, guayusa may be considered a natural source of compounds with potential application in the food and pharmaceutical industries.

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Background: Ilex guayusa Loes. belongs to the family Aquifoliaceae. It is an evergreen tree native to the Amazon region. According to traditional uses, it is used as a diaphoretic, narcotic, purgative, among other uses. Objective: To evaluate the antioxidant capacity, quantify phenols and total flavonoids of extracts obtained from Ilex guayusa leaves. Methods: Total ethanolic extract was obtained using Soxhlet-type equipment, with subsequent liquid/liquid fractionation with solvents of different polarities (petroleum ether, dichloromethane, and ethyl acetate); were reacted with 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+), determining the inhibitory concentration 50 (IC50) and relative antioxidant activity (%AAR), with subsequent statistical analysis (ANOVA and Tukey's post-hoc). Total phenols and flavonoids were quantified using the Folin-Ciocalteu method and complex formation with AlCl3, respectively. Results: The ethanolic extract of leaves showed the highest antioxidant activity in both the DPPH• and ABTS•+ methods with IC50 values of 4.58 and 3.82 ppm, respectively. Conclusions: According to the results obtained in this study, it was possible to corroborate that the ethanolic extract (EE) obtained from the leaves of I. guayusa showed the highest antioxidant capacity, by the DPPH• and ABTS•+ methods. Additionally, it was possible to relate this capacity to the high flavonoids content present in this extract. Based on the above and accompanied by future studies, the Ilex guayusa species could be proposed as an important source of antioxidant compounds with possible application in medicine and the food industry

Antecedentes:Ilex guayusa Loes. pertenece a la familia Aquifoliaceae. Es un árbol perenne, nativo de la región amazónica. De acuerdo con los usos tradicionales es empleada como diaforético, narcótico, purgativo, entre otros usos. Objetivo: Evaluar la capacidad antioxidante, cuantificar fenoles y flavonoides totales de extractos obtenidos a partir de las hojas de Ilex guayusa. Métodos: El extracto etanólico total se obtuvo mediante un equipo tipo "Soxhlet", con posterior fraccionamiento líquido/líquido con solventes de diferentes polaridades (éter de petróleo, diclorometano y acetato de etilo); se hicieron reaccionar con 2,2-difenil-1-picrilhidracilo (DPPH•) y ácido 2,2'-azino-bis-3-etilbenzotiazolin-6-sulfónico (ABTS•+), determinando la concentración inhibitoria 50 (IC50) y la actividad antioxidante relativa (%AAR), con posterior análisis estadístico (ANOVA y post-hoc de Tukey). Los fenoles y flavonoides totales se cuantificaron con el método de Folin-Ciocalteu y el de formación de complejos con AlCl3, respectivamente. Resultados: El extracto etanólico de las hojas fue el que presentó mayor actividad antioxidante tanto en el método DPPH• como en el ABTS•+ con valores de IC50 de 4,58 y 3,82 ppm, respectivamente. Conclusiones: De acuerdo con los resultados obtenidos en este estudio, se pudo corroborar que el extracto etanólico obtenido de las hojas de I. guayusamostró la mayor capacidad antioxidante, por los métodos DPPH• y ABTS•+. Adicionalmente, se pudo relacionar esta capacidad con el alto contenido de flavonoides presentes en este extracto. Con base en lo anterior y acompañado de futuros estudios, la especie I. guayusa podría proponerse como una importante fuente de compuestos antioxidantes con posible aplicación en los campos de la medicina y la industria alimentaria

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The chemical constituents from the leaves of Ilex guayusa were investigated. Sixteen triterpenoids were isolated from the 95% ethanol extract of dried leaves of I. guayusa by silica gel, Sephadex LH-20, and ODS column chromatographies and semi-prepa-rative HPLC. Those triterpenoids were identified by NMR, HR-MS, and literature analysis: 3ß-hydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13ß-olide(1), 3ß-hydroxy-24-nor-4(23),12-oleanadien-28-methyl ester(2), oleanolic acid(3), 3ß,28-dihydroxy-12-oleanene(4), 2α,3ß-dihydroxy-11α,12α-epoxy-24-'nor-olean-4(23)-ene-28,13ß-olide(5), ursolic acid(6), 3ß,23-dihydroxy ursolic acid(7), 3ß,28-dihydroxy-12-ursene(8), 3ß-28-nor-urs-12-ene-3,17-diol(9), 3ß-hydroxyurs-11-ene-28,13ß-olide(10), 13ß,28-epoxy-3ß-hydroxy-11-ursene(11), 3ß-hydroxy-28,28-dimethoxy-12-ursene(12), 3ß-hydroxy-24-nor-urs-4(23),12-dien-28-oic acid(13), 3ß-hydroxy-24-nor-urs-4(23),12-dien-28-methyl ester(14), 2α,3ß-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13ß-olide(15) and 2α,3ß-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23),20(30)-dien-28,13ß-olide(16). Compounds 1-2 were new compounds, and compounds 4-5, 7 and 9-16 were isolated from I. guayusa for the first time.

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A novel functional drink with nutraceutical properties was formulated from the aqueous extracts of Ilex guayusa, and Vernonanthura patens leaves, and cocoa husks. This juice contains various bioactive compounds, such as phenolic compounds and methylxanthines, with antioxidant and stimulant properties of pharmacological interest. However, it is known whether herbal extracts' interaction may have adverse toxic effects on human health. To evaluate this functional drink's innocuity, we estimated the acute oral toxicity (AOT) in experimental mice. This paper presents the AOT evaluation of two formulations of a functional drink (pre-formulation and microencapsulation) at a single dose of 2000 mg/kg of body weight (b.w.). No signs of adverse toxicity and mortality were observed after a single oral dose of 2000 mg/kg b.w. Likewise, no significant body and organ weight changes, food and water consumption behavior, and no histopathological changes were observed in the main organs evaluated. In conclusion, this functional drink can be categorized as low toxicity " according to the Globally Harmonized Classification System (GHS), making it a potential beverage with high nutritional and pharmacological value.

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The chemical constituents from the leaves of Ilex guayusa were investigated. Sixteen triterpenoids were isolated from the 95% ethanol extract of dried leaves of I. guayusa by silica gel, Sephadex LH-20, and ODS column chromatographies and semi-prepa-rative HPLC. Those triterpenoids were identified by NMR, HR-MS, and literature analysis: 3β-hydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13β-olide(1), 3β-hydroxy-24-nor-4(23),12-oleanadien-28-methyl ester(2), oleanolic acid(3), 3β,28-dihydroxy-12-oleanene(4), 2α,3β-dihydroxy-11α,12α-epoxy-24-'nor-olean-4(23)-ene-28,13β-olide(5), ursolic acid(6), 3β,23-dihydroxy ursolic acid(7), 3β,28-dihydroxy-12-ursene(8), 3β-28-nor-urs-12-ene-3,17-diol(9), 3β-hydroxyurs-11-ene-28,13β-olide(10), 13β,28-epoxy-3β-hydroxy-11-ursene(11), 3β-hydroxy-28,28-dimethoxy-12-ursene(12), 3β-hydroxy-24-nor-urs-4(23),12-dien-28-oic acid(13), 3β-hydroxy-24-nor-urs-4(23),12-dien-28-methyl ester(14), 2α,3β-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23)-ene-28,13β-olide(15) and 2α,3β-dihydroxy-11α,12α-epoxy-24-nor-urs-4(23),20(30)-dien-28,13β-olide(16). Compounds 1-2 were new compounds, and compounds 4-5, 7 and 9-16 were isolated from I. guayusa for the first time.

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Due to rapid international market development, there is a strong and urgent need to assess the safety of the novel food, Ilex guayusa. Guayusa has been consumed for centuries in the western Amazon as an herbal tea, and novel food regulation provide a detailed framework for safety assessment of novel foods with such a history of use. This study reviews guayusa's taxonomy, chemical composition, toxicology, ethnobotany, and history of safe use as key elements of a robust novel food safety assessment. Guayusa is a product of traditional agricultural systems with a continuous history of consumption in Ecuador. Its known chemical composition appears to present no greater risk to human health than existing teas such as green tea or yerba mate, although our understanding of guayusa's chemical profile is still nascent, requiring further investigation. Broad consumption of guayusa is not associated with a history of adverse effects or product safety notifications. Biochemical and phytochemical studies have profiled its nutritional content, metabolite composition, and bioactivity, validating guayusa's antioxidant and stimulant properties. In conclusion, guayusa leaves have a well-documented chemical composition and history of safe use, which are key considerations for authorization as a novel food in the EU.

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Ilex species have been consumed traditionally as medicinal teas worldwide. Though the presence of caffeine has been reported in several species, little is known about secondary-metabolite diversity within and among these taxa. Leaf samples of Ilex guayusa, Ilex paraguariensis, and Ilex vomitoria were analyzed by liquid chromatography-mass spectrometry and comparative metabolite profiling with Ilex cassine and other Ilex species to identify chemotaxonomic markers, delimit species, and provide an assessment of chemodiversity. Purine alkaloids were detected and quantified in I. guayusa, I. paraguariensis, and I. vomitoria. Reports of caffeine for I. cassine were not corroborated, suggesting that I. vomitoria was the traditional source of the Native North American tea yaupon. The tetramethyluric acid, theacrine, was detected for the first time in the genus Ilex as a low-level chemotaxonomic marker in I. vomitoria samples. Chemotaxonomy and metabolomics support a close relationship for caffeine-containing Ilex species.

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Unlike all other caffeinated plants, guayusa (Ilex guayusa Loes.) and maté (Ilex paraguariensis A. St. Hill) contain high amounts of pentacyclic triterpenoid acids and alcohols. A phytochemical investigation on these plants revealed a similar triterpenoid profile and a content of ursolic acid (0.7-1%) and amyrin esters (up to 0.5%), quite unusual for dietary plants. The major constituent of the amyrin complex from both plants is α-amyrin palmitate (2a), accompanied by lower amounts of its corresponding palmitoleate (2b) and by the corresponding constitutional isomers from the ß-series (3a and 3b, respectively). Ursolic acid (1) was identified as the responsible for the activity of maté and guayusa extracts in the activation of TGR5, a nuclear receptor of relevance for the prevention and management of diabetes and metabolic syndrome because of its involvement in the regulation of energy expenditure and insulin sensitivity.

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In general, preparations of coffee, teas, and cocoa containing high levels of polyphenols, L-theanine and other bioactive compounds selectively enhance mood and cognition effects of caffeine. This review summarizes the bioactive components of commonly consumed natural caffeine sources (e.g. guayusa, mate and camellia teas, coffee and cocoa) and analyzes the psychopharmacology of constituent phytochemicals: methylxanthines, polyphenols, and L-theanine. Acute and chronic synergistic effects of these compounds on mood and cognition are compared and discussed. Specific sets of constituent compounds such as polyphenols, theobromine and L-theanine appear to enhance mood and cognition effects of caffeine and alleviate negative psychophysiological effects of caffeine. However, more research is needed to identify optimal combinations and ratios of caffeine and phytochemicals for enhancement of cognitive performance.

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BACKGROUND: Guayusa (Ilex guayusa Loes.) leaves, native of the Ecuadorian Amazon, are popularly used for preparing teas. This study aimed to assess the influence of leaf age on the phenolic compounds and carotenoids and the bioactivity and digestibility (in vitro) of aqueous and hydroalcoholic leaf extracts. RESULTS: In total, 14 phenolic compounds were identified and quantified. Chlorogenic acid and quercetin-3-O-hexose were the main representatives of the hydroxycinnamic acids and flavonols respectively. Seven carotenoids were quantified, lutein being the main compound. Ripening affected phenolic content significantly, but there was no significant difference in carotenoid content. Antioxidant capacity, measured by the DPPH• method, was also significantly affected by leaf age. The measurement of in vitro digestibility showed a decrease in phenolic content (59%) as well as antioxidant capacity, measured by the ABTS•+ method, in comparison with initial conditions of the guayusa infusion. Antibacterial and anti-inflammatory activities were assayed with young leaves owing to their higher phenolic contents. Guayusa did not show any antibacterial activity against Escherichia coli ATCC 25922 or Staphylococcus aureus ATCC 25923. Finally, the hydroalcoholic and aqueous extracts exhibited high in vitro anti-inflammatory activity (>65%). CONCLUSION: Young guayusa leaves have potential applications as a functional ingredient in food and pharmaceutical industries. © 2017 Society of Chemical Industry.

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BACKGROUND: Guayusa (Ilex guayusa Loes) is an evergreen tree native of South America that grows particularly in the upper Amazon region of Ecuador. For its health benefits, it has been cultivated and consumed since ancient times by Amazon indigenous tribes. RESULTS: A total of 14 phenolic compounds were identified and quantified. Chlorogenic acid and quercetin-3-O-hexose were the main representatives of the hydroxycinnamic acids and flavonols, respectively. Five carotenoids were identified, showing lutein the highest concentration. Guayusa leaves revealed high antioxidant capacity determined by two analytical methods, DPPH and ORAC. The industrial processing applied to the leaves modified the composition of bioactive compounds and antioxidant capacity of guayusa. In general, blanched guayusa retained the concentration of phenolic compounds and some carotenoids and similar antioxidant capacity as untreated green leaves. In contrast, fermentation reduced the content of bioactive compounds and showed the lowest antioxidant capacity. CONCLUSION: Therefore, blanched guayusa has potential for product development as a functional ingredient in the food industry. © 2017 Society of Chemical Industry.

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Tea from the leaves of guayusa (Ilex guayusa) has a long history of consumption by Ecuadorian natives in regions where the plant is indigenous. The tea contains the methylxanthines caffeine and theobromine as well as chlorogenic acids, flavonoids, and sugars. Various studies were performed to evaluate the general and genetic toxicology of a standardized liquid concentrate of guayusa (GC). Guayusa concentrate was found to be negative in in vitro genotoxicity tests including the Ames test and a chromosome aberration study in human lymphocytes. The oral median lethal dose (LD50) of GC was >5,000 mg/kg for female rats. Guayusa concentrate was administered to male and female rats in a 90-day subchronic study at 1,200, 2,500, and 5,000 mg/kg/d of GC and a caffeine-positive control at 150 mg/kg/d corresponding to the amount of caffeine in the high-dose GC group. Effects observed in the GC-treated groups were comparable to those in the caffeine control group and included reductions in body weights, food efficiency, triglycerides values, and fat pad weights and increases in blood chemistry values for serum aspartate aminotransferase, serum alanine aminotransferase, and cholesterol and adaptive salivary gland hypertrophy. No signs of incremental toxicity due to any other components of guayusa were observed. The studies indicate no harmful effects of GC in these test systems.

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