The Evaluation of Phenolic Acids and Flavonoids Content and Antiprotozoal Activity of Eryngium Species Biomass Produced by Biotechnological Methods

Three species from the Eryngium L. genus—E. campestre, E. maritimum, and E. planum, plants with a rich chemical composition, were selected for phytochemical and biological studies. The applied biotechnological methods allowed to obtain the biomass of these rare or protected species in the form of multiplied shoots (stationary system) and roots cultured in a liquid medium (agitated system). In the extracts from the raw material obtained under in vitro conditions, the content of selected phenolic acids and flavonoids (HPLC-DAD method) as well as the total of polyphenols (Folin–Ciocalteu assay) were quantified. The highest amount of all phenolic compounds was found in extracts from E. planum roots (950.90 ± 33.52 mg/100 g d.w.), and the lowest from E. campestre roots (285.00 ± 10.07 mg/100 g d.w.). The quantitatively dominant compound proved to be rosmarinic acid. The highest amounts were confirmed for E. planum root extract (694.58 mg/100 g d.w.), followed by E. planum (388.95 mg/100 g d.w.) and E. campestre (325.85 mg/100 g d.w.) shoot extracts. The total content of polyphenols was always increased in the biomass from in vitro cultures in comparison to the analogous organs of intact plants of each species. The obtained extracts were assessed for antiprotozoal activity against Acanthamoeba sp. The strength of biological activity of the extracts correlated with the content of phenolic compounds. To our knowledge, this is the first report on the amoebicidal activity of E. campestre, E. maritimum, and E. planum extracts from biomass produced by biotechnological methods.

Inhibitory Effect of Host Ocular Microenvironmental Factors on Chlorhexidine Digluconate Activity

ABSTRACT Acanthamoeba spp. are free-living protozoans that cause a serious human eye disease called Acanthamoeba keratitis (AK). Several new and effective medical therapies for AK patients remain highly debated, and therefore, chlorhexidine digluconate (CHG) is still considered one of the first lines of treatment for AK patients. We hypothesized that ocular microenvironmental factors are responsible for Acanthamoeba drug resistance and clinical AK treatment failure. To investigate the influence of the ocular surface on CHG treatment, we tested the effect of several ocular elements on the antiamoeba activity of CHG. The suspected inhibitory elements, including mucin, albumin, human and amoeba cell lysates, live and heat-killed bacteria, and cornea, were added to the amoebicidal activity platform, where amoebae were incubated with CHG at various concentrations. Mucin showed a significant inhibitory effect on CHG activity against Acanthamoeba castellanii. In contrast, albumin did not affect CHG treatment. Furthermore, human and amoeba cell lysates and live and heat-killed bacterial suspensions also significantly inhibited CHG activity. Additionally, we found that pig corneas also reduced CHG activity. In contrast, dry-eye drops and their major component, propylene glycol, which is commonly used as eyewash material, did not have an impact on CHG activity. Our results demonstrate the effect of ocular microenvironmental factors on CHG activity and suggest that these factors may play a role in the development of amoeba resistance to CHG and treatment failure.

Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani. Additionally, a Microtox® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25–2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

New Protein-Coated Silver Nanoparticles: Characterization, Antitumor and Amoebicidal Activity, Antiproliferative Selectivity, Genotoxicity, and Biocompatibility Evaluation

Nanomaterials quickly evolve to produce safe and effective biomedical alternatives, mainly silver nanoparticles (AgNPs). The AgNPs’ antibacterial, antiviral, and antitumor properties convert them into a recurrent scaffold to produce new treatment options. This work reported the full characterization of a highly biocompatible protein-coated AgNPs formulation and their selective antitumor and amoebicidal activity. The protein-coated AgNPs formulation exhibits a half-inhibitory concentration (IC50) = 19.7 µM (2.3 µg/mL) that is almost 10 times more potent than carboplatin (first-line chemotherapeutic agent) to inhibit the proliferation of the highly aggressive human adenocarcinoma HCT-15. The main death pathway elicited by AgNPs on HCT-15 is apoptosis, which is probably stimulated by reactive oxygen species (ROS) overproduction on mitochondria. A concentration of 111 µM (600 µg/mL) of metallic silver contained in AgNPs produces neither cytotoxic nor genotoxic damage on human peripheral blood lymphocytes. Thus, the AgNPs formulation evaluated in this work improves both the antiproliferative potency on HCT-15 cultures and cytotoxic selectivity ten times more than carboplatin. A similar mechanism is suggested for the antiproliferative activity observed on HM1-IMSS trophozoites (IC50 = 69.2 µM; 7.4 µg/mL). There is no change in cell viability on mice primary cultures of brain, liver, spleen, and kidney exposed to an AgNPs concentration range from 5.5 µM to 5.5 mM (0.6 to 600 µg/mL). The lethal dose was determined following the OECD guideline 420 for Acute Oral Toxicity Assay, obtaining an LD50 = 2618 mg of Ag/Kg body weight. All mice survived the observational period; the histopathology and biochemical analysis show no differences compared with the negative control group. In summary, all results from toxicological evaluation suggest a Category 5 (practically nontoxic) of the Globally Harmonized System of Classification and Labelling of Chemicals for that protein-coated AgNPs after oral administration for a short period and urge the completion of its preclinical toxicological profile. These findings open new opportunities in the development of selective, safe, and effective AgNPs formulations for the treatment of cancer and parasitic diseases with a significant reduction of side effects.