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BACKGROUND: Extensive antifungal drug use has enhanced fungal resistance, resulting in persistent mycoses. Combining antifungal plant extracts/compounds with these drugs offers good alternatives to increase the activity of both partners, minimize side effects, and overcome drug resistance. In our previous study, Phytolacca tetramera berries extracts demonstrated activity against Candida spp., correlating with the amount of the main constituent phytolaccoside B and its genin, phytolaccagenin. The extracts and phytolaccagenin altered the fungal plasma membrane by binding to ergosterol, whereas phytolaccoside B increased chitin synthase activity. However, the presence of triterpenoid saponins in Phytolacca spp. has been linked to acute toxicity in humans. PURPOSE: This study aimed to evaluate combinations of P. tetramera berries extracts, phytolaccoside B and phytolaccagenin, together with commercial antifungals [amphotericin B, fluconazole, itraconazole, posaconazole, and caspofungin] against Candida albicans and Candida glabrata, to find synergistic effects with multi-target actions, in which the doses of both partners are reduced, and therefore their toxicity. Additionally, we intended to explore their anti-virulence capacity, thereby hindering the development of drug-resistant strains. METHODS: The effects of these combinations were evaluated using both the checkerboard and isobologram methods. Fractional Inhibitory Concentration Index and Dose Reduction Index were calculated to interpret the combination results. To confirm the multi-target effect, studies on mechanisms of action of synergistic mixtures were performed using ergosterol-binding and quantification assays. The ability to inhibit Candida virulence factors, including biofilm formation and eradication from inert surfaces, was also evaluated. Quantification of active markers was performed using a validated UHPLC-ESI-MS method. RESULTS: Eight synergistic combinations of P. tetramera extracts or phytolaccagenin (but not phytolaccoside B) with itraconazole or posaconazole were obtained against C. albicans, including a resistant strain. These mixtures acted by binding to ergosterol, decreasing its whole content, and inhibiting Candida biofilm formation in 96-well microplates and feeding tubes in vitro, but were unable to eradicate preformed biofilms. CONCLUSIONS: This study demonstrated the synergistic and anti-virulence effects of P. tetramera berries extracts and phytolaccagenin with antifungal drugs against Candida spp., providing novel treatment avenues for fungal infections with reduced doses of both natural products and commercial antifungals, thereby mitigating potential human toxicity concerns.

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Plant species Phytolacca americana (Phytolaccaceae) can be classified as UFP (Unconventional Food Plant); it is used for food and/or medicinal purposes due to its analgesic, anti-inflammatory, anti-rheumatic and anti-arthritic properties; moreover, it is suitable to treat different skin diseases. However, its seeds present integumentary dormancy caused by uneven germination. The aim of the present study is to assess the effect of chemical scarification in sulfuric acid on dormancy overcome in Phytolacca Americana (Phytolaccaceae) seeds. Seeds were immersed in 4 ml sulfuric acid, which was concentrated through different immersion times (2, 5, 8, 11 and 15 min), and compared to the control treatment (without sulfuric acid). The study followed a completely randomized design with six treatments and four repetitions, with 50 seeds per treatment. The following parameters were assessed: days to start germination (DSG), germination (G), germination speed index (GSI), abnormal plantlets (AP), dead seeds (DS), hard seeds (HS) and total dry mass (TDM). Data were subjected to Shapiro-Wilk test for variance normality. The analysis of variance and regression were carried out in RStudio software, version 4.1.0. Means recorded for treatments were compared through Tukey test (p ≤ 0.05). The treatment based on immersion in sulfuric acid for 2 min was the most efficient in overcoming dormancy in this species, since it led to the best results for germination variables and total biomass accumulation in plantlets.

A Phytolacca americana (Phytolaccaceae) é uma espécie que pode ser classificada como PANC (Planta Alimentícia Não Convencional), utilizada para fins alimentícios e/ou medicinais com propriedades analgésicas, antiinflamatórias, antirreumáticas, antiartríticas e, também é adequada para o tratamento de várias doenças de pele. Contudo, suas sementes apresentam dormência tegumentar causando desuniformidade na germinação. Objetivou-se com o presente trabalho avaliar o efeito da escarificação química com ácido sulfúrico na superação da dormência em sementes de Phytolacca americana (Phytolaccaceae). As sementes foram imersas em 4 ml de ácido sulfúrico concentrado por diferentes períodos de imersão (2, 5, 8, 11 e 15 min) comparadas ao tratamento controle (sem ácido sulfúrico). O delineamento utilizado foi o inteiramente casualizado com seis tratamentos e quatro repetições de 50 sementes por tratamento. Foram avaliados os seguintes parâmetros: dias para iniciar a germinação (DIG), germinação (G), índice de velocidade de germinação (IVG), plântulas anormais (PA), sementes mortas (SM), sementes duras (SD), e massa seca total (MST). Os dados foram submetidos ao teste de Shapiro-Wilk para normalidade das variâncias. E a análise de variância e de regressão por meio do software RStudio versão 4.1.0. Sendo as médias dos tratamentos comparadas pelo teste Tukey (p ≤ 0,05). O tratamento de imersão em ácido sulfúrico por 2 min mostrou-se o mais eficiente na superação da dormência dessa espécie, proporcionando melhores resultados nas variáveis germinativas e acúmulo de biomassa total das plântulas.

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PURPOSE: The present randomized controlled clinical trial evaluated the efficacy of homeopathic medicines of Melissa officinalis (MO), Phytolacca decandra (PD), and the combination of both in the treatment of possible sleep bruxism (SB) in children. STUDY DESIGN: Patients (n = 52) (6.62 ± 1.79 years old) were selected based on the parents report of SB. The study comprised a crossover design that included 4 phases of 30-day treatment (Placebo; MO 12c; PD 12c; and MO 12c + PD 12c), with a wash-out period of 15 days between treatments. METHODS: At baseline and after each phase, the Visual Analogic Scale (VAS) was used as the primary outcome measure to evaluate the influence of treatments on the reduction of SB. The following additional outcome measures were used: a children's sleep diary with parent's/guardian's perceptions of their children's sleep quality, the trait of anxiety scale (TAS) to identify changes in children's anxiety profile, and side effects reports. Data were analyzed by ANOVA with repeated measures followed by Post Hoc LSD test. RESULTS: Significant reduction of SB was observed in VAS after the use of Placebo (-1.72 ± 0.29), MO (-2.36 ± 0.36), PD (-1.44 ± 0.28) and MO + PD (-2.21 ± 0.30) compared to baseline (4.91 ± 1.87). MO showed better results compared to PD (p = 0.018) and Placebo (p = 0.050), and similar result compared to MO+PD (p = 0.724). The sleep diary results and TAS results were not influenced by any of the treatments. No side effects were observed after treatments. CONCLUSION: MO showed promising results in the treatment of possible sleep bruxism in children, while the association of PD did not improve MO results.

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BACKGROUND: Phytolacca tetramera is an endemic plant from Argentina that is currently at serious risk because its environment is subjected to a high anthropic impact. A previous study has shown that berry extracts obtained from this plant display antifungal activity against multiple human-pathogenic fungi when tested with a non-standardized method. Further evidences of the antifungal properties of other parts of the plant and studies of mechanism of antifungal action of the antifungal chemically characterized extracts are required. PURPOSE: This study aimed to gain further evidence of the antifungal activity of P. tetramera berry, leaf and root extracts in order to find the most active extract to be developed as an Herbal Medicinal Antifungal Product. The medicinal usefulness of P. tetramera extracts as antifungal agents will serve as an important support to create concience and carry out actions tending to the preservation of this threatened species and its environment. MATERIALS AND METHODS: Chemical analysis of all P. tetramera extracts, including quantitation of selected markers, was performed through UHPLC-ESI-MS/MS and UPLC-ESI-MS techniques according to the European Medicines Agency (EMA). The antifungal activity of the quantified extracts was tested with the standardized CLSI microbroth dilution method against Candida spp. Antifungal mechanisms of the most active extract were studied by examination of morphological changes by phase-contrast and fluorescence microscopies and both, cellular and enzymatic assays targeting either the fungal membrane or the cell wall. RESULTS: The antifungal activity of twelve P. tetramera extracts was tested against Candida albicans and Candida glabrata. The dichloromethane extract from berries (PtDEb) showed the best activity. Phytolaccagenin (PhytG) and phytolaccoside B (PhytB) were selected as the main active markers for the antifungal P. tetramera extracts. The quantitation of these active markers in all extracts showed that PtDEb possessed the highest amount of PhytG and PhytB. Finally, studies on the mechanism of antifungal action showed that the most active PtDEb extract produces morphological changes compatible with a damage of the cell wall and/or the plasma membrane. Cellular and enzymatic assays showed that PtDEb would not damage the fungal cell wall by itself, but would alter the plasma membrane. In agreement, PtDEb was found to bind to ergosterol, the main sterol of the fungal plasma membrane. CONCLUSION: Studies of the anti-Candida activity of P. tetramera extracts led to the selection of PtDEb as the most suitable extract, confirming the antifungal properties of the threatened species P. tetramera. The new data give a valuable reason for the definitive protection of this sp. and its natural environment thus allowing further studies for the future development of an Herbal Medicinal Antifungal Product.

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Besides the known compounds ( ± ) 3,3-bis-demethylpinoresinol (2), americanol A (3), spergulagenic acid (4), epi-acetylaleuritolic acid (5), 6'-palmityl-α-spinateryl-d-glucoside (6a) and 6'-palmityl-δ(7)-stigmastenyl-d-glucoside (6b), a novel peltogynoid (1) named icosandrin was obtained from the dried fruits of Phytolacca icosandra. This new compound was characterised by 1D-/2D-NMR, UV, IR and HR-MS techniques as 11ξ-methoxy-6,7-methylenedioxy-[2]benzopyrano-[4,3-b][1]-benzopyran-4-one. Toxicity of 1 was assessed through the Brine Shrimp Lethality Assay. Lignan 2 is reported for the first time in Phytolaccaceae family.

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The in vivo anthelmintic (AH) activity of the ethanolic extract from leaves of Phytolacca icosandra was evaluated in goats artificially infected with Haemonchus contortus. Parasite naïve goats were artificially infected with 3000 H. contortus infective larvae per animal. Once the infection was patent (day 28 post-infection) all the animals were sampled to determine the faecal egg counts (FEC) for five consecutive days. Two groups of animals were formed balanced for their FEC and body-weight (BW) (n=6/group): the non-treated control group and the treated group in which goats were individually administered with the ethanolic extract of P. icosandra. The extract was administered orally using gelatin capsules (250 mg/kg BW) which were dosed on two consecutive days using a pill-dispenser. Faecal samples were collected from each animal from the day of dosage (Day 0) on a daily basis to determine the number of eggs per gram of feces (EPG) for 15 days post-treatment (PT). The FEC of the two groups were compared using the repeated measures analyses of variance using the log transformed data Ln (FEC+1). The presence of saponins, coumarins, flavonoids, steroids and terpenoids were detected by standard methodologies in the extract. The P. icosandra ethanolic extract was further analyzed by gas chromatography (GC) coupled to a mass spectrometry (GC-MS). A significant reduction in FEC was observed in the treated group compared to the control from day 7 until day 15 PT (P<0.05). The highest percentage reduction (72%) was found on day 11 PT. No adverse reactions were observed in all treated animals for the entire trial. The GC-MS analysis of the organic extracts revealed the presence of three fatty acids as compounds with highest abundance. The three compounds that were identified by their mass fragmentation patterns were: 2-Pentadecanone, 6, 10, 14-trimethyl (RT 10.3 min), Pentadecanoic acid, 14-methyl-, methyl ester (RT 10.8 min) and Hexadecanoic acid, ethyl ester (RT 11.2 min). It is concluded that the P. icosandra ethanolic extract obtained from leaves showed in vivo anthelmintic activity against H. contortus when administered orally to goats at a dose of 250 mg/kg BW on two consecutive days. The dose used did not cause any negative effects on the health of goats.

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The development of anthelmintic resistance has impacted on the success of conventional anthelmintics (AH) for the control of gastrointestinal nematodes in grazing/browsing sheep and goats. Medicinal plants from the traditional herbolary in Mexico may provide new candidates that can be explored as alternative sources of AHs for ruminants. This study evaluated the leaf extracts derived from Phytolacca icosandra against infective L(3) larvae and eggs from Haemonchus contortus collected from sheep. Three extracts of different polarities were obtained from the leaf plants using ethanol, n-hexane and dichloromethane as the solvents. The effectiveness of the in vitro AH activity of the plant extracts was evaluated using larval migration inhibition (LMI) and egg hatch (EHA) assays. For the LMI assays, the ethanolic extract of P. icosandra showed 55.4% inhibition of larval migration at 2mg/mL (p<0.05). The dichloromethane extract of P. icosandra showed 67.1% inhibition of migration at 3mg/mL (p<0.05) and a dose-dependent response with an LD(50) of 0.90 mg/mL. The n-hexane extract failed to show inhibition of larval migration at any concentration explored. In the EHA for the ethanol extract, the lowest concentration tested (0.15 mg/mL) resulted in inhibition of egg hatching greater than 72.6%. Therefore, the LD(50) could not be calculated for this extract. The LD(50) of the dichloromethane extract of P. icosandra was 0. 28 mg/mL. An egg hatch inhibition greater than 90% was observed with both the ethanolic and dichloromethane extracts when using a concentration of 0.90 mg/mL or higher. The n-hexane extract failed to show egg hatch inhibition at any concentration tested. The AH activity reported for P. icosandra could be attributable to the flavonoids, steroids, terpenoids, coumarins and/or saponins that were present in the ethanolic and dichloromethane extracts. A combination of more than one component may also explain the observed AH activity against the H. contortus life stages that were evaluated. In conclusion, the ethanolic and dichloromethane extracts of P. icosandra showed clear in vitro AH activity against the H. contortus eggs and the L(3) larvae. However, the hexanic extract of the plant leaves failed to show any in vitro AH activity.

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A saponin-rich extract of Phytolacca dioica L. berries, its acid hydrolysate, and its major aglycone, phytolaccagenin, were assayed for antifungal activity against ATCC standard cultures of Candida albicans and Cryptococcus neoformans, and against clinical isolates of these fungi. The activity of the extract was either low or negligible, but the hydrolysate, containing the sapogenins, including phytolaccagenin, and also pure phytolaccagenin, showed promising antifungal potency. Hydrolysis of a natural product extract is shown to be a useful modification leading to improved bioactivity.

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Four known serjanic acid glycosides were isolated from the fruits of Phytolacca rugosa and characterized mainly by 2D NMR spectroscopy and mass spectrometry. This aglycon has a chemotaxonomic significance for the genus Phytolacca.

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Through the action of glycosyltransferases, a plant can biosynthetically assemble small different aglycons or 'templates' to various polysaccharides to produce numerous glycoconjugates differing in the type of the attached aglycon, the anomeric configuration of C-1 of the glycosylating sugar, the type of sugar and the different position of attachments of the sugar unit present in the polysaccharide chain. The position of attachments and the anomeric configuration of the different sugar present in the polysaccharide create the opportunity to generate molecules with either the same or very close molecular weights, which have relative structural similarity--forming isobaric and positional isomers. Although isomeric differentiation was once considered outside of the domain of mass spectrometry, this task can now be resolved using tandem mass spectrometry. In a standardized purified glycoconjugate fraction (SPT01) from Phytolacca bogotensis, we report conventional electrospray ionization mass spectrometry and collision-induced dissociation (CID) MS/MS parameters which favored the formation of characteristic product ions. This allowed us to suggest the type of sugar linkages present in a specific glycoconjugate. Ten new glycoconjugate are described from this plant and another twelve known saponins were structurally characterized using the automatic MSn acquisition mode. The differentiation of two pairs of positional isomers and four isobaric glycosides and the production of a library of 30 glycosides present in P. bogotensis were accomplished.

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