Limonium Brasiliense Research Articles

Limonium brasiliense rhizomes extract against virulence factors of Porphyromonas gingivalis: Inhibition of gingipains, bacterial adhesion, and biofilm formation

Periodontitis is clinically characterized by destruction of the tooth support system and tooth loss. Porphyromonas gingivalis (Pg) plays a dominant role in periodontitis. Fractions and isolated compounds from an acetone-water extract of the roots of Limonium brasiliense (Lb) were tested in vitro for their anti-adhesive capacity against Pg on human KB buccal cells, influence on gingipains, the main virulence factors of Pg, and biofilm formation. Fractions EAF and FLB7 (50 μg/mL) reduced the bacterial adhesion of Pg to KB cells significantly (63 resp. 70%). The proanthocyanidin samarangenin A inhibited the adhesion (72%, 30 μM), samarangenin B (71%, 20 μM), and the flavan-3-ol epigallocatechin-3-O-gallate (79%, 30 μM). Fraction AQF, representing hydrophilic compounds, reduced the proteolytic activity of Arginin-specific gingipain (IC50 12.78 μg/mL). Fractions EAF and FLB7, characterized by lipohilic constituents, inhibited Arg-gingipain (IC50 3 μg/mL). On Lysine-specific gingipain, AQF has an IC50 15.89, EAF 14.15, and FLB7 6 μg/mL. The reduced bacterial adhesion is due to a strong interaction of proanthocyanidins with gingipains. AQF, EAF, and FLB7 significantly inhibited biofilm formation: IC50 11.34 (AQF), 11.66 (EAF), and 12.09 μg/mL (FLB7). In silico analysis indicated, that the polyphenols act against specific targets of Pg, not affecting mammalian cells. Therefore, Lb might be effective for prevention of periodontal disease by influencing virulence factors of Pg.

Towards a comprehensive understanding of zinc tolerance in Limonium brasiliense as a useful tool for environmental remediation and monitoring.

Heavy metal pollution is a serious environmental problem worldwide, creating the necessity to find eco-friendly strategies for monitoring and remediating environments. This study aimed to evaluate morphological, physiological, and biochemical responses as indicative of Zn tolerance in Limonium brasiliense and to determine the ability of this halophyte to accumulate different concentrations of Zn (0, 100, and 200 μM) in hydroponic conditions. The leaf shape at high Zn concentration showed enlarged petioles and lanceolate blades, whereas the leaf size was reduced. Water content, chlorophyll fluorescence parameters, and pigment content decreased with Zn addition. Of the antioxidant activities, only APx increased 75% compared to the control by Zn stress. Zn concentration was higher in aerial structures than in roots (BAC>1 and TF>1), suggesting that L. brasiliense could function as an accumulator of Zn. Its great ability to resist metal stress and its strong capacity to protect itself against high Zn concentration postulate it as a good phytoremediation of environments enriched with Zn. The study emphasizes using leaf morphology as an early biomonitoring tool for detecting Zn pollution, providing more evidence of their potential use as a biomarker for evaluating and assessing ecosystem health in biomonitoring programs.

Assessment of halophyte plant phenotypic responses under heavy metals pollution. Implications for monitoring and phytoremediation

While phytoremediation is a highly valued practice to address local pollution problems, the use of early biomarkers of stress is useful for monitoring environments since they allow us to take measures before deleterious effects are irreversible. In this framework the goals are: to evaluate the pattern of leaf shape variation of Limonium brasiliense plants related to a metal soil gradient in the San Antonio salt marsh; to assess whether seeds from sites with different pollution levels show the same pattern of leaf shape variations under optimal growing conditions; and to compare the growth, the Pb accumulation pattern, and the leaf shape variation pattern of plants germinated from seeds originated in sites with different pollution levels in response to an experimental Pb rise. The results obtained from leaves collected in the field showed that the leaf shape changed depending on the soil metal levels. Plants germinated from seeds collected at the different sites expressed all the variation in leaf shape independently of the origin site, and the mean shape of each site was close to the consensus. Instead, when looking for the leaf shape components that maximize the differences between the sites from a growth experiment exposed to an increase in Pb in the irrigation solution, the pattern of variation found in the field disappeared. That is, only plants from the polluted site did not show variations in leaf shape in response to Pb additions. Finally, Pb accumulation in the roots was highest in plants germinated from seeds from the site where the soil pollution is greater. That suggests that seeds of L. brasiliense from polluted sites are better to use in phytoremediation practices, specifically to stabilize Pb in its roots whilst plants from the non-polluted site are better to detect pollutant soils using the leaf shape as an early biomarker.

Microstructured Polymer System Containing Proanthocyanidin-Enriched Extract from Limonium brasiliense as a Prophylaxis Strategy to Prevent Recurrence of Porphyromonas gingivalis.

Periodontal diseases are a global oral health problem affecting almost 10% of the global population. Porphyromonas gingivalis is one of the main bacteria involved in the initiation and progression of inflammatory processes as a result of the action of the cysteine proteases lysin- and arginine-gingipain. Surelease/polycarbophil microparticles containing a lyophilized proanthocyanidin-enriched fraction from the rhizomes of Limonium brasiliense, traditionally named "baicuru" (ethyl acetate fraction), were manufactured. The ethyl acetate fraction was characterized by UHPLC by the presence of samarangenins A and B (12.10 ± 0.07 and 21.05 ± 0.44%, respectively) and epigallocatechin-3-O-gallate (13.44 ± 0.27%). Physiochemical aspects of Surelease/polycarbophil microparticles were characterized concerning particle size, zeta potential, entrapment efficiency, ethyl acetate fraction release, and mucoadhesion. Additionally, the presence of the ethyl acetate fraction-loaded microparticles was performed concerning potential influence on viability of human buccal KB cells, P. gingivalis adhesion to KB cells, gingipain activity, and P. gingivalis biofilm formation. In general, all Surelease/polycarbophil microparticles tested showed strong adhesion to porcine cheek mucosa (93.1 ± 4.2% in a 30-min test), associated with a prolonged release of the ethyl acetate fraction (up to 16.5 ± 0.8% in 24 h). Preincubation of KB cells with Surelease/polycarbophil microparticles (25 µg/mL) resulted in an up to 93 ± 2% reduced infection rate by P. gingivalis. Decreased activity of the P. gingivalis-specific virulence factors lysin- and arginine-gingipain proteases by Surelease/polycarbophil microparticles was confirmed. Surelease/polycarbophil microparticles decreased biofilm formation of P. gingivalis (97 ± 2% at 60 µg/mL). Results from this study prove the promising activity of Surelease/polycarbophil microparticles containing ethyl acetate fraction microparticles as a prophylaxis strategy to prevent the recurrence of P. gingivalis.

Multidimensional approach to evaluate Limonium brasiliense as source of early biomarkers for lead pollution monitoring under different saline conditions

• The effect of Pb and salinity in Limonium brasiliense was experimentally studied. • Pb and salt rises have a negative impact on growth. • Pb and salt rises induce leaf shape variations and antioxidant system responses. • The translocation of Pb from roots to shoots is limited in L. brasiliense . • This work is a milestone for studying Pb and salt effects in Patagonian saltmarsh plants. The search for early indicators of stress (biomarkers) with low cost of determination (both economic and logistic) is a prominent issue in environmental studies. Consequently, an experimental study was conducted with the aim of validating the leaf shape variations as an early biomarker in plants growing in contaminated soils. We manipulated the Pb (0, 45 and 90 µM) and salt (0 and 598 mM NaCl) concentrations to evaluate their accumulation pattern, and their effect in biomass, leaf shape, photosynthetic pigments, phenolic contents, antioxidant enzyme activities, and lipid peroxidation in Limonium brasiliense . Results showed that growth and chlorophylls pigments synthesis in L. brasiliense are mainly impacted by Pb rise. Furthermore, the activities of the antioxidant enzymes and the lipid peroxidation both were increased by the Pb and salt addition; whereas phenolic content appeared to be more sensitive to salinity rise. On the other hand, changes in leaf shape were induced with Pb principally, and salt rise. The plants growing under stress conditions (Pb and salt treatments) showed expanded petioles and lanceolate limbos. Pb concentrations were always higher in roots than in aerial structures, suggesting that L. brasiliense could be a good candidate to the phytostabilization of Pb in polluted soils. The agreement in the changes of the several response variables measured as a product of the manipulated stressful factors suggests that L. brasiliense is a suitable bioindicator and their phenotypic plasticity is an early biomarker of stress by contamination. Leaf shape resulted to be a useful, practical and low-cost biomarker of stress on plants growing on polluted environmental conditions.

In vitro evaluation of the protective effects of plant extracts against amyloid-beta peptide-induced toxicity in human neuroblastoma SH-SY5Y cells.

Alzheimer’s disease (AD) is the most common form of dementia and has no cure. Therapeutic strategies focusing on the reduction of oxidative stress, modulation of amyloid-beta (Aβ) toxicity and inhibition of tau protein hyperphosphorylation are warranted to avoid the development and progression of AD. The aim of this study was to screen the crude extracts (CEs) and ethyl-acetate fractions (EAFs) of Guazuma ulmifolia, Limonium brasiliense, Paullinia cupana, Poincianella pluviosa, Stryphnodendron adstringens and Trichilia catigua using preliminary in vitro bioassays (acetylcholinesterase inhibition, antioxidant activity and total polyphenol content) to select extracts/fractions and assess their protective effects against Aβ25–35 toxicity in SH-SY5Y cells. The effect of the EAF of S. adstringens on mitochondrial membrane potential, lipid peroxidation, superoxide production and mRNA expression of 10 genes related to AD was also evaluated and the electropherogram fingerprints of EAFs were established by capillary electrophoresis. Chemometric tools were used to correlate the in vitro activities of the samples with their potential to be evaluated against AD and to divide extracts/fractions into four clusters. Pretreatment with the EAFs grouped in cluster 1 (S. adstringens, P. pluviosa and L. brasiliense) protected SH-SY5Y cells from Aβ25-35-induced toxicity. The EAF of S. adstringens at 15.62 μg/mL was able completely to inhibit the mitochondrial depolarization (69%), superoxide production (49%) and Aβ25-35-induced lipid peroxidation (35%). With respect to mRNA expression, the EAF of S. adstringens also prevented the MAPT mRNA overexpression (expression ratio of 2.387x) induced by Aβ25–35, which may be related to tau protein hyperphosphorylation. This is the first time that the neuroprotective effects of these fractions have been demonstrated and that the electropherogram fingerprints for the EAFs of G. ulmifolia, L. brasiliense, P. cupana, P. pluviosa and S. adstringens have been established. The study expands knowledge of the in vitro protective effects and quality control of the evaluated fractions.

Morpho-anatomical characters of Limonium brasiliense leaves

Limonium brasiliense (Boiss.) Kuntze, Plumbaginaceae, is a coastal herb of the Southern Brazilian states and Rio de Janeiro State. In folk medicine, it is used in the treatment of female genitourinary tract infections and menstrual cycle irregularities. The aim of this study was to describe morpho-anatomical characters with details on venation pattern of L. brasiliense leaves, collected on the coast of Rio Grande do Sul State, in Southern Brazil. Leaf samples fixed in FAA50 (5% formaldehyde, 5% acetic acid and 90% ethanol: water mixture 50:50 v/v) and 1% glutaraldehyde and preserved in 70% ethanol, were histologically sectioned and analyzed by light and scanning electron microscopy. The L. brasiliense leaves were simple, entire, incomplete, oboval-lanceolate, venation penninervous and showed rosulated phyllotaxy, entire and revolute margin, with apex rounded to slightly retuse, base attenuate and venation pattern brochidodromous. On microscopic analysis, these leaves showed a thick and striated cuticle, salt glands, isobilateral mesophyll thicker in the apical region of the leaf blade, abundant aerenchyma in the petiole and main vein, collateral vascular bundles surrounded by fiber-sclereids, lipid substances in the cuticle and polyphenols in phloem cells.

Soil-geomorphology relationships and landscape evolution in a southwestern Atlantic tidal salt marsh in Patagonia, Argentina

Salt marshes in Patagonia ecosystem are nowadays fully recognized by ecological, pollution and phytoremediation studies but a soil genesis and geomorphology approach is currently unknown. The aim of this study was to establish the soil-geomorphology relationship in Fracasso salt marsh and to determine the successional vegetation dynamics associated with the landscape evolution. This work was carried out in Fracasso salt marsh sited in Península Valdés, Argentina, where an integrated study on soil-geomorphology relationship and landscape evolution was performed along with sedimentological analysis and vegetation changes (C3 photosynthesis pathway vs. C4 photosynthesis pathway plants). This last was determined through the δ13C composition from soil organic matter (SOM). Soil descriptions and laboratory analysis of soil samples were performed. A marked relationship between the vegetation unit, the dominant landform and the type of associated soil was found. Limonium brasiliense (Lb) and Sarcocornia perennis (Sp), both C3 plants, are dominant in levees associated with tidal creeks, and soils were classified as Typic Fluvaquents, while Spartina alterniflora (Sa) soils were classified as Sodic Endoaquents and Sodic Psammaquents. Although no sulfidic materials were identified by incubation test, they were identified by hydrogen peroxide treatment in Sa soils, and now are considered potential acid sulfate soils (PASS). Sedimentological analysis from deepest sandy C horizons indicates a beach depositional environment. On the other hand, the δ13C stable isotope composition of SOM preserved into these buried soil acting as parent materials shows the dominance of C4 plants presumably belonging to Spartina species, suggesting a possible colonization and stabilization as the pioneer salt marsh.

Flavan-3-ols and proanthocyanidins from Limonium brasiliense inhibit the adhesion of Porphyromonas gingivalis to epithelial host cells by interaction with gingipains

Porphyromonas gingivalis is a pathogen strongly involved in chronic and aggressive forms of periodontitis. Natural products, mainly polyphenols, have been described for advanced treatment of periodontitis by inhibition of the bacterial adhesion of P. gingivalis to the epithelial host cells. An acetone:water extract (LBE) from the rhizomes of Limonium brasiliense (Boiss.) Kuntze was tested under in vitro conditions for potential antiadhesive effects against P. gingivalis to human KB cells and for inhibition of the proteolytic activity of gingipains, the main virulence factor of P. gingivalis. LBE≤100μg/mL had no cytotoxicity against the bacteria and did not influence the cell physiology of human epithelial KB cells. At 100μg/mL LBE reduced the adhesion of P. gingivalis to KB cells significantly by about 80%. LBE at 20μg/mL reduced the proteolytic activity of the arginin-specific Rgp gingipain by about 75%. Chemical profiling of LBE indicated the presence of gallic acid, epigallocatechin-3-O-gallate and samarangenins A and B as lead compounds. UHPLC by using MS and UV detection displays a suitable method for quality control of the extract for identification and quantification of the lead compounds.

Pharmacognostic evaluation, and development and validation of a HPLC-DAD technique for gallocatechin and epigallocatechin in rhizomes from Limonium brasiliense

Limonium brasiliense (Boiss.) Kuntze, Plumbaginaceae, is a plant from the southern coast of Brazilian that is used for the treatment of premenstrual syndrome, menstrual disorders and genito-urinary infections. The aim of the present study was to determine the quality control parameters for rhizomes collected during different periods by pharmacopoeial and non-pharmacopoeial methods, and to develop and validate a HPLC-DAD method for quantitative control of marker substances. The measured parameters were: granulometric analysis (d50=0.21–0.48mm), loss on drying (11.1–12.4%), total ash (4.9–5.7%), dry residue by extraction with acetone:water (7:3, v/v) (30.6–39.5%), total polyphenol content (8.5–15.8%), and chromatographic fingerprint by HPLC and TLC. Besides, the acetone:water (7:3, v/v) extraction solvent in combination with a turbo-extractor, yielded the crude extract with a significant increase in tannins (F4,20=37.0, p<0.001). The antioxidant potential of the crude acetone:water (7:3, v/v) extract, as well as the ethyl acetate and water fractions obtained after the partition process was evaluated by DPPH and the results were, respectively: IC50 6.87, 5.91, and 6.92μg/ml. The validation parameters for the HPLC-DAD method showed adequate specificity, precision and accuracy. The gallo- and epigallocatechin contents were, respectively, 0.8–2.7% and 1.2–2.2%. These data contribute to analysis of the pharmacognostic quality control of the commonly used part from this species.

Acute toxicity and long-term safety evaluation of the crude extract from rhizomes of Limonium brasiliense in mice and rats

Ethnopharmacological relevanceLimonium brasiliense (Boiss.) Kuntze, Plumbaginaceae, popularly known as baicuru, has been used in folk medicine to treat menstrual cramps and to regulate menstrual periods. However, little is known about its safety. This study evaluated the safety through in vivo tests of the acute, long-term, and liver toxicity, and the mutagenicity of the crude extract (CE) from rhizomes of L. brasiliense. Materials and methodsThe acute toxicity was assessed in Swiss mice, and the chronic toxicity in Wistar rats. Male and female mice received the CE orally in single doses of 1.0, 2.0, 3.0, 4.0, or 5.0g/kg. Clinical changes and mortality rate were used as parameters to assess the acute toxicity. In the long-term evaluation, male and female Wistar rats were treated orally with daily doses of the CE (50, 100, or 200mg/kg) for 90 days. Assessments of weight, behavior and food intake, urinalysis, biochemical and hematological analyses, as well as macro- and microscopic observations of several organs were performed. The redox state of the liver was evaluated as a means of investigating the liver toxicity, and the micronucleus test to assess mutagenicity was also performed. ResultsEvaluation of acute toxicity indicated no apparent clinical change in the animals; the LD50 was 4.8g/kg. Evaluation after 90 days administration showed that the CE, even in higher doses than are considered therapeutic, appeared to be safe. The micronucleus test demonstrated a low mutagenic potential for the CE. ConclusionOur results showed that treatment with the CE from L. brasiliense caused low or no toxicity, as assessed using these doses and evaluation methods.

Morpho-anatomical study of rhizome of Limonium brasiliense

Limonium brasiliense (Boiss.) Kuntze, Plumbaginaceae, is an herb popularly known as guaicuru, guaicurá or baicuru. The species inhabits salt marshes from the coastal region of southern Brazil, including Rio de Janeiro, to Uruguay and Argentina. Although widely used in folk medicine in the state of Rio Grande do Sul to treat genitourinary infections and to regulate menstrual periods, L. brasiliense has been little studied. The present morpho-anatomical study was undertaken to resolve some doubts in the literature as to the nature of the part of the plant that is used for medicinal purposes, a true rhizome or a root. The morpho-anatomical characteristics were analyzed with the aid of light and scanning electron microscopy. The botanical material was characterized as a rhizome with internodes that are evident in the younger but not the older portions. Microscopic analysis revealed the presence of a multilayered periderm with a cortex, ray parenchyma, and pith, formed by collenchyma tissue with abundant intercellular spaces in the outer portions of the cortex, responsible for the rigidity of the body, and with walls impregnated with phenolic compounds. The vascular bundles are collateral with elliptical to elongated cells, and with few conducting and sclerenchymal elements. Groups of sclereids are dispersed through the cortex and pith. These morpho-anatomical characteristics define the structure as a rhizome.

Application and Analysis of the Folin Ciocalteu Method for the Determination of the Total Phenolic Content from Limonium Brasiliense L.

Limonium brasiliense is a common plant on the southern coast of Brazil. The roots are traditionally used for treatment of premenstrual syndrome, menstrual disturbances and genito-urinary infections. Pharmaceutical preparations obtained from its roots and used for these purposes were marketed in Brazil in the 1980s and 1990s. Currently, the Brazilian Drug Agency (National Health Surveillance Agency, ANVISA) has canceled the registration of these products, and their use was discontinued because of a lack of studies to characterize the plant raw material and ensure the effectiveness and safety of its use. The aim of the present study was to develop and validate an analytical method to determine the content of total polyphenols (TP) in an extract from L. brasiliense roots, by the UV/Vis spectrophotometric method. L. brasiliense roots were extracted in acetone:water (7:3, v/v-10% w/v). The crude extract was used to develop a method for TP assay. The method was validated according to national and international guidelines. The optimum conditions for analysis time, wavelength, and standard substance were 30 min, 760 nm, and pyrogallol, respectively. Under these conditions, validation by UV/Vis spectrophotometry proved the method to be linear, specific, precise, accurate, reproducible, robust, and easy to perform. This methodology complies with the requirements for analytical application and to ensure the reliability of the results.

Patterns of substrata use by the invasive acorn barnacle Balanus glandula in Patagonian salt marshes

Balanus glandula is a native barnacle of the rocky shores in the west coast of North America. Forty years after its introduction in Argentina, this species is the only barnacle dominating the high intertidal of local rocky shores and more interesting, it was also reported successfully colonizing soft-bottom salt marshes. In this study, we identified and characterized the substrata most successfully colonized by B. glandula in Patagonian salt marshes through descriptive and experimental means. We surveyed and compared two Patagonian salt marshes and the substrata colonized by B. glandula. Our results show that barnacles utilize more than 10 types of different substrata. Mussel valves were the most frequent type of substrata utilized in Riacho marsh, whereas the dominant halophyte Sarcocornia perennis was the substratum most utilized in Fracasso marsh where mussels were mostly absent. When the five most utilized substrata were experimentally offered, the halophyte shrub Limonium brasiliense was the most densely colonized of the experimental substrata, with the largest sizes and the lowest proportion of dead barnacles. Density and size of the barnacles recruited on plants were similar to that observed in nearby invaded rocky shores. Our study strongly suggests that soft-bottom environments, where hard substrata are available, have to be seriously considered when designing early detection plans targeting B. glandula and other similar rocky shore invasive species.

Antioxidant Metabolites from Limonium brasiliense (Boiss.) Kuntze

A free radical scavenging activity guided fractionation of the polar extract from roots of Limonium brasiliense (Plumbaginaceae) led to the isolation of five active compounds including: myricetin 3-O-alpha-rhamnopyranoside (1), (-)-epigallocatechin 3-O-gallate (2), (-)-epigallocatechin (3), (+)-gallocatechin (4) and gallic acid (5). These and other chemical constituents are reported for the first time for this species. The characterization of these compounds was achieved by spectroscopic methods (1H NMR, 13C NMR and UV).