Ação antifúngica de extratos de plantas isolados sobre espécies de Candida albicans e Candida não-albicans / Antifungal action of isolated plant extracts on Candida albicans and Candida non-albicans species

As espécies de Candida spp. apresentam-se como o principal patógeno fúngico humano, podendo causar infecções superficiais e invasivas. A emergência de novas espécies em infecções, apresentando alta resistência aos antifúngicos utilizados desafia pesquisadores a propor novas terapias no controle desta infecção, entre as quais podemos citar a fitoterapia realizando o uso de extratos de plantas para propor novos protocolos. Por isto, este trabalho objetiva avaliar a ação antifúngica dos extratos isolados de Quilaia (Quillaja saponaria) e Alcachofra (Cynara scolymus) sobre C. albicans, C. glabrata, C. krusei, C. tropicalis e C. dubliniensis em formas planctônica e biofilmes monotípicos. Inicialmente foram feitas análises da ação antifúngica dos extratos de Quilaia e Alcachofra por meio do teste de microdiluição em caldo (CLSI ­ Protocolo M27-S4), para determinar as Concentrações Inibitórias Mínimas (CIM) e as Concentrações Fungicidas Mínimas (CFM) de espécies. Os biofilmes foram formados por 48 h em poços de microplacas, os quais receberam tratamentos de concentrações dos extratos (100 mg/mL, 50 mg/mL, 25 mg/mL, 12,5 mg/mL e 6,25 mg/mL), assim como foram testados os grupos controles positivo e negativo, para determinação da viabilidade celular por meio do teste MTT. Os dados foram analisados estatísticamente pelos testes ANOVA e Tukey, com significância de 5%. Os resultados da CIM e CFM para as espécies C. albicans, C. krusei e C. glabrata foram de 12,5mg/mL para ambos os extratos, os valores para C tropicalis foi 12,5 mg/mL para o extrato de Quilaia e 25 mg/mL para Alcachofra, ambos os extratos apresentaram o mesmo valor de 6,25 mg/mL para a espécie C. dubliniensis. A ação antibiofilme do extrato de Quilaia apresentou redução fúngica do biofilme principalmente nas duas maiores concentrações (100 mg/mL e 50 mg/mL) do extrato para ambos os tempos (5 min e 24 h) quando comparados com o grupo controle negativo que não recebeu tratamento, apresentando diferenças estatísticas significativas (p<0.001). A ação antibiofilme do extrato de Alcachofra apresentou reduções dos biofilmes significativas nas cinco concentrações (100 mg/mL, 50 mg/mL, 25 mg/mL, 12,5 mg/mL e 6,25 mg/mL) em ambos os tempos, na maioria das espécies, apresentando diferenças significativas (p<0.001). Diante disso, concluímos que os extratos glicólicos de Q. saponaria e C. scolymus apresentam ação antifúngica em todas as espécies de Candida spp. analisadas, sendo um potencial antifúngico para C. albicans e as espécies C. não-albicans, mas na espécie de C. krusei as reduções de biofilme só ocorrem nas maiores concentrações. Os resultados da ação antibiofilme manteve um padrão de ação, quanto maior a concentração do extrato, maior a redução, isto para ambos os extratos e para a maioria das espécies analisadas (AU)

Candida spp. They are the main human fungal pathogen and can cause superficial and invasive infections. The emergence of new species in infections, presenting high resistance to the antifungals used, challenges researchers to propose new therapies to control this infection, among which we can mention phytotherapy using plant extracts to propose new protocols. Therefore, this work aims to evaluate the antifungal action of extracts isolated from Quilaia (Quillaja saponaria) and Artichoke (Cynara scolymus) on C. albicans, C. glabrata, C. krusei, C. tropicalis and C. dubliniensis in planktonic forms and biofilms monotypic. Initially, analyzes of the antifungal action of Quilaia and Artichoke extracts were carried out using the broth microdilution test (CLSI ­ Protocol M27-S4), to determine the Minimum Inhibitory Concentrations (MICs) and Minimum Fungicide Concentrations (MFCs) of species. Biofilms were formed for 48 h in microplate wells, which received extract concentration treatments (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL), as well as the positive and negative control groups were tested to determine cell viability using the MTT test. The data were statistically analyzed using the ANOVA and Tukey tests, with a significance of 5%. The MIC and CFM results for the species C. albicans, C. krusei and C. glabrata were 12.5 mg/mL for both extracts, the values for C tropicalis were 12.5 mg/mL for the Quilaia extract and 25 mg/mL for Artichoke, both extracts presented the same value of 6.25 mg/mL for the species C. dubliniensis. The antibiofilm action of the Quilaia extract showed a fungal reduction of the biofilm mainly at the two highest concentrations (100 mg/mL and 50 mg/mL) of the extract for both times (5 min and 24 h) when compared with the negative control group that did not receive treatment, showing significant statistical differences (p<0.001). The antibiofilm action of Artichoke extract showed significant reductions in biofilms at the five concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL) at both times, in most species, showing significant differences (p<0.001). Therefore, we conclude that glycolic extracts of Q. saponaria and C. scolymus have antifungal action on all species of Candida spp. analyzed, with antifungal potential for C. albicans and non-albicans C. species, but in the C. krusei species, biofilm reductions only occur at higher concentrations. The results of the antibiofilm action maintained a pattern of action, the higher the concentration of the extract, the greater the reduction, this for both extracts and for the majority of species analyzed(AU)

Quillaja brasiliensis nanoparticle adjuvant formulation improves the efficacy of an inactivated trivalent influenza vaccine in mice.

The threat of viral influenza infections has sparked research efforts to develop vaccines that can induce broadly protective immunity with safe adjuvants that trigger robust immune responses. Here, we demonstrate that subcutaneous or intranasal delivery of a seasonal trivalent influenza vaccine (TIV) adjuvanted with the Quillaja brasiliensis saponin-based nanoparticle (IMXQB) increases the potency of TIV. The adjuvanted vaccine (TIV-IMXQB) elicited high levels of IgG2a and IgG1 antibodies with virus-neutralizing capacity and improved serum hemagglutination inhibition titers. The cellular immune response induced by TIV-IMXQB suggests the presence of a mixed Th1/Th2 cytokine profile, antibody-secreting cells (ASCs) skewed toward an IgG2a phenotype, a positive delayed-type hypersensitivity (DTH) response, and effector CD4+ and CD8+ T cells. After challenge, viral titers in the lungs were significantly lower in animals receiving TIV-IMXQB than in those inoculated with TIV alone. Most notably, mice vaccinated intranasally with TIV-IMXQB and challenged with a lethal dose of influenza virus were fully protected against weight loss and lung virus replication, with no mortality, whereas, among animals vaccinated with TIV alone, the mortality rate was 75%. These findings demonstrate that TIV-IMXQB improved the immune responses to TIV, and, unlike the commercial vaccine, conferred full protection against influenza challenge.

A Bioinformatics Tool for Efficient Retrieval of High-Confidence Terpene Synthases (TPS) and Application to the Identification of TPS in Coffea and Quillaja.

Terpenoids are a class of compounds that are found in all living organisms. In plants, some terpenoids are part of primary metabolism, but most terpenes found in plants are classified as specialized metabolites, encoded by terpene synthases (TPS). It is not obvious how to assign the putative product of a given TPS using bioinformatics tools. Phylogenetic analyses easily assign TPS into families; however members of the same TPS family can synthetize more than one terpenoid-and, in many biotechnological applications, researchers are more interested in the product of a given TPS rather than its phylogenetic profile. Automated protein annotation can be used to classify TPS based on their products, despite the family they belong to. Here, we implement an automated bioinformatics method, search_TPS, to identify TPS proteins that synthesize mono, sesqui and diterpenes in Angiosperms. We verified the applicability of the method by classifying wet lab validated TPS and applying it to find TPS proteins in Coffea arabica, C. canephora, C. eugenioides, and Quillaja saponaria. Search_TPS is a computational tool based on PERL scripts that carries out a series of HMMER searches against a curated database of TPS profile hidden Markov models. The tool is freely available at https://github.com/liliane-sntn/TPS .

Soapbark Triterpenes: Quillaja brasiliensis Cell Culture Sapogenin and Free Sterol Analysis by GCMS.

Triterpene saponins of the genus Quillaja (Quillajaceae) are known for their immunoadjuvant, hypocholesterolemic, and anti-inflammatory activity. Plant cell cultures are useful for the study of saponin metabolism and industrial production of these bioactive compounds. While structurally related phytosterols are primary metabolites essential to growth and development, saponins are responsive to pathogen and abiotic stress, fulfilling roles in plant specialized metabolism. For cell culture production of saponins, phytosterols may be considered a competing pathway which relies on a common pool of cytosolic isoprenoid precursors.Understanding the metabolic allocation of resources between these two related pathways is key to maximizing saponin production in in vitro production systems. Sterols and saponins naturally occur in multiple conjugated forms, which complicate separation and quantification. The acid hydrolysis of conjugated sterols and saponins to their free forms is a useful technique to simplify their analysis by gas chromatography. Here we provide the workflow for the quantification of free sterols and sapogenins in cell cultures of Quillaja brasiliensis .

Formulation of IMXQB: Nanoparticles Based on Quillaja brasiliensis Saponins to be Used as Vaccine Adjuvants.

Adjuvants are essential components of subunit, recombinant, nonreplicating and killed vaccines, as they are substances that boost, shape, and/or enhance the immune response triggered by vaccination. Saponins obtained from the Chilean Q. saponaria tree are used as vaccine adjuvants in commercial vaccines, although they are scarce and difficult to obtain. In addition, tree felling is needed during its extraction, which has ecological impact. Q. brasiliensis leaf-extracted saponins arise as a more sustainable alternative, although its use is still limited to preclinical studies. Despite the remarkable immunostimulating properties of saponins, they are toxic to mammalian cells, due to their intrinsic characteristics. For these reasons they are mostly used in veterinary vaccines, although recently the Q. saponaria purified saponin QS-21 has been included in adjuvant systems for human vaccines, such as Mosquirix and Shingrix (GSK). In order to abrogate the toxicity of the saponins fractions, they can be formulated as immunostimulating complexes (ISCOMs). ISCOM-matrices are cage-like nanoparticles of approximately 40 nm, formulated combining saponins and lipids, without antigen, and are great adjuvants able to promote Th1-biased immune responses in a safe manner. Herein we describe how to formulate ISCOM-matrices nanoparticles using Q. brasiliensis purified saponin fractions (IMXQB) by the dialysis method. In addition, we indicate how to verify the appropriate size and homogeneity of the formulated nanoparticles.

Structure Elucidation of Triterpenoid Saponins Found in an Immunoadjuvant Preparation of Quillaja brasiliensis Using Mass Spectrometry and 1H and 13C NMR Spectroscopy.

An immunoadjuvant preparation (named Fraction B) was obtained from the aqueous extract of Quillaja brasiliensis leaves, and further fractionated by consecutive separations with silica flash MPLC and reverse phase HPLC. Two compounds were isolated, and their structures elucidated using a combination of NMR spectroscopy and mass spectrometry. One of these compounds is a previously undescribed triterpene saponin (Qb1), which is an isomer of QS-21, the unique adjuvant saponin employed in human vaccines. The other compound is a triterpene saponin previously isolated from Quillaja saponaria bark, known as S13. The structure of Qb1 consists of a quillaic acid residue substituted with a ß-d-Galp-(1→2)-[ß-d-Xylp-(1→3)]-ß-d-GlcpA trisaccharide at C3, and a ß-d-Xylp-(1→4)-α-l-Rhap-(1→2)-[α-l-Arap-(1→3)]-ß-d-Fucp moiety at C28. The oligosaccharide at C28 was further substituted at O4 of the fucosyl residue with an acyl group capped with a ß-d-Xylp residue.

Zika Virus Envelope Domain III Recombinant Protein Delivered With Saponin-Based Nanoadjuvant From Quillaja brasiliensis Enhances Anti-Zika Immune Responses, Including Neutralizing Antibodies and Splenocyte Proliferation.

Nanoadjuvants that combine immunostimulatory properties and delivery systems reportedly bestow major improvements on the efficacy of recombinant, protein-based vaccines. Among these, self-assembled micellar formulations named ISCOMs (immune stimulating complexes) show a great ability to trigger powerful immunological responses against infectious pathogens. Here, a nanoadjuvant preparation, based on saponins from Quillaja brasiliensis, was evaluated together with an experimental Zika virus (ZIKV) vaccine (IQB80-zEDIII) and compared to an equivalent vaccine with alum as the standard adjuvant. The preparations were administered to mice in two doses (on days zero and 14) and immune responses were evaluated on day 28 post-priming. Serum levels of anti-Zika virus IgG, IgG1, IgG2b, IgG2c, IgG3 were significantly increased by the nanoadjuvant vaccine, compared to the mice that received the alum-adjuvanted vaccine or the unadjuvanted vaccine. In addition, a robust production of neutralizing antibodies and in vitro splenocyte proliferative responses were observed in mice immunized with IQB80-zEDIII nanoformulated vaccine. Therefore, the IQB80-zEDIII recombinant preparation seems to be a suitable candidate vaccine for ZIKV. Overall, this study identified saponin-based delivery systems as an adequate adjuvant for recombinant ZIKV vaccines and has important implications for recombinant protein-based vaccine formulations against other flaviviruses and possibly enveloped viruses.

IMXQB-80: A Quillaja brasiliensis saponin-based nanoadjuvant enhances Zika virus specific immune responses in mice.

Vaccine adjuvants are compounds that enhance/prolong the immune response to a co-administered antigen. Saponins have been widely used as adjuvants for many years in several vaccines - especially for intracellular pathogens - including the recent and somewhat revolutionary malaria and shingles vaccines. In view of the immunoadjuvant potential of Q. brasiliensis saponins, the present study aimed to characterize the QB-80 saponin-rich fraction and a nanoadjuvant prepared with QB-80 and lipids (IMXQB-80). In addition, the performance of such adjuvants was examined in experimental inactivated vaccines against Zika virus (ZIKV). Analysis of QB-80 by DI-ESI-ToF by negative ion electrospray revealed over 29 saponins that could be assigned to known structures existing in their congener Q. saponaria, including the well-studied QS-21 and QS-7. The QB-80 saponins were a micrOTOF able to self-assembly with lipids in ISCOM-like nanoparticles with diameters of approximately 43 nm, here named IMXQB-80. Toxicity assays revealed that QB-80 saponins did present some haemolytical and cytotoxic potentials; however, these were abrogated in IMXQB-80 nanoparticles. Regarding the adjuvant activity, QB-80 and IMXQB-80 significantly enhanced serum levels of anti-Zika virus IgG and subtypes (IgG1, IgG2b, IgG2c) as well as neutralized antibodies when compared to an unadjuvanted vaccine. Furthermore, the nanoadjuvant IMXQB-80 was as effective as QB-80 in stimulating immune responses, yet requiring fourfold less saponins to induce the equivalent stimuli, and with less toxicity. These findings reveal that the saponin fraction QB-80, and particularly the IMXQB-80 nanoadjuvant, are safe and capable of potentializing immune responses when used as adjuvants in experimental ZIKV vaccines.

A Two-Step Orthogonal Chromatographic Process for Purifying the Molecular Adjuvant QS-21 with High Purity and Yield.

QS-21 is a triterpene glycoside saponin found in the bark of the Chilean soap bark tree Quillaja saponaria. It is a highly potent vaccine adjuvant that is included in two approved vaccines and has shown promise in numerous other vaccine candidates in the research and clinical pipelines. One major hurdle to the widespread use of this adjuvant is the difficulty of obtaining it in high yield and purity. Previously reported purification approaches either showed suboptimal purity and/or yield, lacked efficiency, or had strict requirement on the composition of the starting material. Here, we report the development of a new two-step orthogonal chromatographic process, consisting of a polar reversed-phase (RP) chromatography step followed by a hydrophilic interaction chromatography (HILIC) step, for purifying QS-21 from a commercially available Quillaja saponaria bark extract with high yield and > 97% purity. This process makes available a simple and efficient method for obtaining highly pure QS-21 from saponin-enriched bark extract.

Saponin production from Quillaja genus species. An insight into its applications and biology

Quillaja genus (Quillajaceae family) is endemic to South America, where is represented by two species, Quillaja saponaria and Quillaja brasiliensis. One outstanding characteristic of these forest tree species is their production of saponins, a family of amphipathic glycosides, involved in the defensive response of plants against biotic and abiotic factors. Saponins are metabolites of economic importance due to their chemical and physical properties. Basic and applied research efforts performed during the last decades, mainly on Q. saponaria, have placed these compounds as an important raw material in industrial areas, such as food and beverage, cosmetics, vaccine production, biopesticides, among others. In this review, we summarize information on saponins from Quillaja species during the last years, analyzing current developments by application areas, as well as their chemical composition and properties. We also describe the general advances in revealing saponin biosynthesis pathways, related genes and Quillaja genomes, as well as the conservation status, domestication processes, and perspectives in the context of implementing genetic improvement programs.

Saponin production from Quillaja genus species. An insight into its applications and biology

Quillaja genus (Quillajaceae family) is endemic to South America, where is represented by two species, Quillaja saponaria and Quillaja brasiliensis. One outstanding characteristic of these forest tree species is their production of saponins, a family of amphipathic glycosides, involved in the defensive response of plants against biotic and abiotic factors. Saponins are metabolites of economic importance due to their chemical and physical properties. Basic and applied research efforts performed during the last decades, mainly on Q. saponaria, have placed these compounds as an important raw material in industrial areas, such as food and beverage, cosmetics, vaccine production, biopesticides, among others. In this review, we summarize information on saponins from Quillaja species during the last years, analyzing current developments by application areas, as well as their chemical composition and properties. We also describe the general advances in revealing saponin biosynthesis pathways, related genes and Quillaja genomes, as well as the conservation status, domestication processes, and perspectives in the context of implementing genetic improvement programs.(AU)

In vitro evaluation and molecular docking of QS-21 and quillaic acid from Quillaja saponaria Molina as gastric cancer agents.

The cytotoxic mechanism of the saponin QS-21 and its aglycone quillaic acid (QA) was studied on human gastric cancer cells (SNU1 and KATO III). Both compounds showed in vitro cytotoxic activity with IC50 values: 7.1 µM (QS-21) and 13.6 µM (QA) on SNU1 cells; 7.4 µM (QS-21) and 67 µM (QA) on KATO III cells. QS-21 and QA induce apoptosis on SNU1 and KATO III, as demonstrated by TUNEL, Annexin-V and Caspase Assays. Additionally, we performed in silico docking studies simulating the binding of both triterpenic compounds to key proteins involved in apoptotic pathways. The binding energies (∆Gbin) thus calculated, suggest that the pro-apoptotic protein Bid might be a plausible target involved in the apoptotic effect of both triterpenic compounds. Although QA shows some antiproliferative effects on SNU1 cells cultured in vitro, our results suggest that QS-21 is a more powerful antitumor agent, which merits further investigation regarding their properties as potential therapeutic agents for gastric cancer.

A liquid chromatography coupled to quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS) method for identification analysis of saponins from Quillaja saponaria bark extracts in foot-and-mouth disease vaccines: Development, validation and applicability.

Saponins from Quillaja saponaria have been commonly used as immunomodulatory adjuvants in foot-and-mouth disease vaccines (FMDVs). However, due to the lack of consensus over the possible exacerbation of local inflammatory responses in cattle and its economic impacts, their use has been discouraged by Brazilian authorities. A qualitative method intended to determine the presence of saponins from Q. saponaria bark extracts in FMDVs was developed and validated. Instrumental analysis was performed using an liquid chromatography (LC) coupled to a quadrupole-time-of-flight-mass spectrometry (TOF-MS) system. The method was validated according to the International Conference on Harmonization Harmonized Tripartite Guideline Q2 (R1) and Brazilian Ministry of Agriculture, Livestock and Food Supply Analytical Quality Assurance Guidelines. Validation parameters were determined and considered suitable to the established criteria. The validated method has been applied in routine analysis in the National Agricultural Laboratory at Rio Grande do Sul (LANAGRO-RS). All results obtained were in agreement with the vaccine's composition described by the manufacturer. The method is easy and adequate for analysis in routine laboratories. To the best of the authors' knowledge, this is the first report of a method which intends to investigate the presence of saponins from Q. saponaria bark extracts in veterinary vaccines.

Quillaja bark saponin effects on Kluyveromyces lactis ß-galactosidase activity and structure.

Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis ß-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin-protein interaction. Circular dichroism shows that saponin changes the ß-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of ß-galactosidase with the artificial substrate o-nitrophenyl-ß-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.

Foamy matters: an update on Quillaja saponins and their use as immunoadjuvants.

Immunoadjuvant Quillaja spp. tree saponins stimulate both cellular and humoral responses, significantly widening vaccine target pathogen spectra. Host toxicity of specific saponins, fractions and extracts may be rather low and further reduced using lipid-based delivery systems. Saponins contain a hydrophobic central aglycone decorated with several sugar residues, posing a challenge for viable chemical synthesis. These, however, may provide simpler analogs. Saponin chemistry affords characteristic interactions with cell membranes, which are essential for its mechanism of action. Natural sources include Quillaja saponaria barks and, more recently, Quillaja brasiliensis leaves. Sustainable large-scale supply can use young plants grown in clonal gardens and elicitation treatments. Quillaja genomic studies will most likely buttress future synthetic biology-based saponin production efforts.

Antihyperalgesic Activity of Quillaic Acid Obtained from Quillaja Saponaria Mol.

BACKGROUND: Quillaja saponaria Mol. bark contains a high concentration of triterpene saponins that have been used for centuries as a cleansing, antiinflammatory and analgesic agent in Chilean folk medicine. In earlier studies, in mice, both the anti-inflammatory as well as the antinociceptive effect of the major sapogenin, quillaic acid have been demonstrated (QA). OBJECTIVE: To determine the antihyperalgesic effect of QA one and seven days after itpl administration of complete Freund's adjuvant (CFA) in male mice using the hot plate test in the presence of complete Freund's adjuvant (HP/CFA) as an acute and chronic skeletal muscle pain model. METHODS: The present study evaluated the antihyperalgesic activity of QA against acute and chronic skeletal muscle pain models in mice using the hot plate test in the presence of complete Freund's adjuvant (HP/CFA), at 24 h (acute assay) and 7 days (chronic assay) , with dexketoprofen (DEX) as the reference drug. RESULTS: In acute and chronic skeletal muscle pain assays, QA at 30 mg/kg ip elicited its maximal antihyperalgesic effects (65.0% and 53.4%) at 24 h and 7 days, respectively. The maximal effect of DEX (99.0 and 94.1 at 24 h and 7 days, respectively) was induced at 100 mg/kg. CONCLUSION: QA and DEX elicit dose-dependent antihyperalgesic effects against acute and chronic skeletal muscle pain, but QA is more potent than DEX in the early and late periods of inflammatory pain induced by CFA.

Structural characterisation of new immunoadjuvant saponins from leaves and the first study of saponins from the bark of Quillaja brasiliensis by liquid chromatography electrospray ionisation ion trap mass spectrometry.

INTRODUCTION: Quillaja brasiliensis (St. A. -Hil. & Tul) Mart (Quillajaceae) is a species native to South America, which is rich in saponins. Saponins are used in different industries, so there is a constant demand for this type of compound. Based on the wide range of applications for the saponins found in this species, notably as immunoadjuvants, we conducted a comprehensive study of this tree and its saponins. OBJECTIVE: The purpose of this work is to complete the characterisation of the immunoadjuvant saponin fraction from Q. brasiliensis leaves and further study the saponin fraction obtained from Q. brasiliensis bark. METHODOLOGY: Saponin fractions were studied using mass spectrometry in combination with classical methods of monosaccharide and methylation analysis. We performed direct infusion and liquid chromatography/electrospray ionisation ion trap multiple-stage mass spectrometry (DI-ESI-IT-MSn and LC-ESI-IT-MS2 ). RESULTS: Seventy-five saponins, 21 from leaves and 54 from bark, were tentatively identified according to their molecular mass, fragmentation pattern and chromatographic behaviour. This work represents the first investigation of saponins from the bark of Q. brasiliensis and some of them presented new structural motifs not previously reported in the genus Quillaja. CONCLUSION: The efficiency and selectivity of the data dependent LC-MS2 method allowed the rapid profiling of saponins from Q. brasiliensis. The results of the monosaccharide and methylation analysis performed in saponins from Q. brasiliensis fractions and Q. saponaria Molina (Quillajaceae) fraction gives further support to the structures proposed according to the mass spectral data, validating the strategy used in the present work.

Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities.

Quillaja saponaria Molina represents the main source of saponins for industrial applications. Q. saponaria triterpenoids have been studied for more than four decades and their relevance is due to their biological activities, especially as a vaccine adjuvant and immunostimulant, which have led to important research in the field of vaccine development. These saponins, alone or incorporated into immunostimulating complexes (ISCOMs), are able to modulate immunity by increasing antigen uptake, stimulating cytotoxic T lymphocyte production (Th1) and cytokines (Th2) in response to different antigens. Furthermore, antiviral, antifungal, antibacterial, antiparasitic, and antitumor activities are also reported as important biological properties of Quillaja triterpenoids. Recently, other saponins from Q. brasiliensis (A. St.-Hill. & Tul.) Mart. were successfully tested and showed similar chemical and biological properties to those of Q. saponaria barks. The aim of this manuscript is to summarize the current advances in phytochemical and pharmacological knowledge of saponins from Quillaja plants, including the particular chemical characteristics of these triterpenoids. The potential applications of Quillaja saponins to stimulate further drug discovery research will be provided.

Germination rates of four Chilean forest trees seeds: Quillaja saponaria, Prosopis chilensis, Vachellia caven, and Caesalpinia spinosa.

Data on the germination rates of four tree species, natively founded in the Chilean Mediterranean-climate zone, were determined by germination in crop chambers. The obtained data were used to interpolate or extrapolate the time taken for 50% of seeds to germinate in each case. These results are useful for regional native forest research and, in a broad sense, for its use in models to study germination dynamics in Mediterranean-climate zones.

Quillaja brasiliensis saponin-based nanoparticulate adjuvants are capable of triggering early immune responses.

Commercially available saponins are extracted from Quillaja saponaria barks, being Quil A® the most widely used. Nanoparticulate immunostimulating complexes (ISCOMs or ISCOMATRIX) formulated with these, are able to stimulate strong humoral and cellular immune responses. Recently, we formulated novel ISCOMs replacing QuilA® by QB-90 (IQB-90), a Quillaja brasiliensis leaf-extracted saponin fraction, and reported that IQB-90 improved antigen uptake, and induced systemic and mucosal antibody production, and T-cell responses. However, its mechanism of action remains unclear. In this study we provide a deeper insight into the immune stimulatory properties of QB-90 and ISCOMATRIX-like based on this fraction (IMXQB-90). We show herein that, when used as a viral vaccine adjuvant, QB-90 promotes an "immunocompetent environment". In addition, QB-90 and IMXQB-90 induce immune-cells recruitment at draining-lymph nodes and spleen. Subsequently, we prove that QB-90 or IMXQB-90 stimulated dendritic cells secret IL-1ß by mechanisms involving Caspase-1/11 and MyD88 pathways, implying canonical inflammasome activation. Finally, both formulations induce a change in the expression of cytokines and chemokines coding genes, many of which are up-regulated. Findings reported here provide important insights into the molecular and cellular mechanisms underlying the adjuvant activity of Q. brasiliensis leaf-saponins and its respective nanoparticles.