Comparison of DNA extraction methods for COVID-19 host genetics studies.

The coronavirus disease 2019 (COVID-19) pandemic has resulted in global shortages in supplies for diagnostic tests, especially in the developing world. Risk factors for COVID-19 severity include pre-existing comorbidities, older age and male sex, but other variables are likely play a role in disease outcome. There is indeed increasing evidence that supports the role of host genetics in the predisposition to COVID-19 outcomes. The identification of genetic factors associated with the course of SARS-CoV-2 infections relies on DNA extraction methods. This study compared three DNA extraction methods (Chelex®100 resin, phenol-chloroform and the QIAamp DNA extraction kit) for COVID-19 host genetic studies using nasopharyngeal samples from patients. The methods were compared regarding number of required steps for execution, sample handling time, quality and quantity of the extracted material and application in genetic studies. The Chelex®100 method was found to be cheapest (33 and 13 times cheaper than the commercial kit and phenol-chloroform, respectively), give the highest DNA yield (306 and 69 times higher than the commercial kit and phenol-chloroform, respectively), with the least handling steps while providing adequate DNA quality for downstream applications. Together, our results show that the Chelex®100 resin is an inexpensive, safe, simple, fast, and suitable method for DNA extraction of nasopharyngeal samples from COVID-19 patients for genetics studies. This is particularly relevant in developing countries where cost and handling are critical steps in material processing.

Chemical composition, pesticidal activities and in-silico investigation of Hedychium spicatum Sm. chloroform extract.

The present study aimed to identify the bioactive constituents in the chloroform extract of H. spicatum rhizomes (HS-RCLE), further evaluated for its in-vitro pesticidal activities validating via molecular docking techniques. GC/MS analysis of HS-RCLE identified 14 compounds contributing 84.1 % of the total composition. The extract was dominated by oxygenated sesquiterpenes (43.1 %) with curcumenone (25.2 %) and coronarin E (14.8 %) as the major compounds. The extract recorded 89.4 % egg hatchability inhibition and 82.6 % immobility of Meloidogyne incognita, 66.7 % insecticidal activity on Spodoptera litura, 100 % phytotoxic activity on Raphanus raphanistrum seeds, and 74.7 % anti-fungal activity on Curvularia lunata at the respective highest dose studied. The biological activities were furthermore validated by using docking studies on certain proteins/enzymes namely acetylcholinesterase (PBD ID: IC2O), carboxylesterase (PDB ID: 1CI8), acetohydroxyacid synthase (PBD ID: 1YHZ) and trihydroxy naphthalene reductase (PBD ID: 3HNR). The bioactivity of the major constituents of the extract was predicted with the help of in silico PASS studies. HS-RCLE was observed to be a viable alternative source of natural pesticidal agents and paves the way for further studies on its mechanistic approaches and field trials to ascertain its pesticidal studies.

Investigation of antibacterial and antifungal activity of Saussurea costus root extracts.

Infectious diseases are a serious danger to public health, and plants may be a potential source of novel antimicrobial agents. In this study, the antibacterial and antifungal activity of the essential oil, hexane-chloroform, methanolic, and aqueous extracts of Saussurea costus (S. costus) root were evaluated against Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterobacter cloacae, Enterococcus faecalis, Klebsiella pneumonia, Acinetobacter baumannii, Escherichia coli, and Candida albicans. For this evaluation, disc diffusion and micro- dilution susceptibility assays were performed. Chemical analysis was also performed to determine phytochemical constituents of the extracts. Our results showed that the essential oil and methanolic extract of S. costus root exhibited the highest antimicrobial activity, followed by hexane-chloroform extract, with aqueous extract showing the lowest activity. The highest activity with the lowest MIC value was recorded as 3.12 µl/ml for the essential oil (against S. epidermidis and C. albicans), 3.12 mg/ml for the methanolic extract (against S. aureus), and 6.25 mg/ml for both hexane-chloroform and aqueous extracts (against S. aureus). In general, the tested extracts had moderate to good antimicrobial activity against the tested gram-positive bacteria and C. albicans. S. costus root can be considered as a potential natural source of antimicrobial agents to fight pathogen microorganisms.

The anti-aging, anti-tuberculosis and antioxidant potential benefits of Saudi Arabia Olea-Europaea Leaves extracts.

The olive leaf extract and olive leaf indicated a high potential for application in food additives and foodstuffs. It could be these bio-products useful and important in condition therapy related with oxidative stress and can use it to develop functional foods and to improve the food's shelf life. The olive leaf chemical composition of Oleaeuropaea L. grown from eljouf in Saudi Arabia, using solvents of increasing polarity cyclohexane, dichloromethane, chloroform, ethyl acetate, methanol and ethanol was determined using by GC/MS. Furthermore, the antioxidant activity (diphenylpicrylhydrazyl (DPPH), anti-aging, and anti-tuberculosis of olive leaf extracts were evaluated. The results indicated that extract of Oleaeuropaea L. has a considerable contains in polyphenols (hydroxytyrosol, oleuropein and their derivatives) regarding its antioxidant effects, the major components were detected by GC/MS in Olea dichloromethane extract are Hexadecanoic acid (15.82%), 7(4Dimethylaminophenyl)3,3,12trimethyl3,12dihydro6 Hpyrano[2,3c]acridin 6 one (11.21%), and in Olea chloroform extract are Hexatriacontane (12.68%), nTetratr iacontane (10.95%). The results concluded that the plant extract of chloroform showed no anti-aging activities and the lower anti-aging activities for cyclohexane extract, while, the Olea dichloromethane extract was the most active extract. The obtained data confirmed that the most active extract of anti-tubercolisis was for chloroform and ethyl acetate extract, while, anti-tubercolisis activity of ethanolic extract was the lower. The extract amount as well as the solvent polarity influence the inhibitory activity. A favorable connection was demonstrated inter alia the leaf extracts antioxidant activity and the content of total phenol.

Bio-Prospecting of Crude Leaf Extracts from Thirteen Plants of Brazilian Cerrado Biome on Human Glioma Cell Lines.

(1) Background: Malignant gliomas are aggressive tumors characterized by fast cellular growth and highly invasive properties. Despite all biological and clinical advances in therapy, the standard treatment remains essentially palliative. Therefore, searching for alternative therapies that minimize adverse symptoms and improve glioblastoma patients' outcomes is imperative. Natural products represent an essential source in the discovery of such new drugs. Plants from the cerrado biome have been receiving increased attention due to the presence of secondary metabolites with significant therapeutic potential. (2) Aim: This study provides data on the cytotoxic potential of 13 leaf extracts obtained from plants of 5 families (Anacardiaceae, Annonaceae, Fabaceae, Melastomataceae e Siparunaceae) found in the Brazilian cerrado biome on a panel of 5 glioma cell lines and one normal astrocyte. (3) Methods: The effect of crude extracts on cell viability was evaluated by MTS assay. Mass spectrometry (ESI FT-ICR MS) was performed to identify the secondary metabolites classes presented in the crude extracts and partitions. (4) Results: Our results revealed the cytotoxic potential of Melastomataceae species Miconia cuspidata, Miconia albicans, and Miconia chamissois. Additionally, comparing the four partitions obtained from M. chamissois crude extract indicates that the chloroform partition had the greatest cytotoxic activity against the glioma cell lines. The partitions also showed a mean IC50 close to chemotherapy, temozolomide; nevertheless, lower toxicity against normal astrocytes. Analysis of secondary metabolites classes presented in these crude extracts and partitions indicates the presence of phenolic compounds. (5) Conclusions: These findings highlight M. chamissois chloroform partition as a promising component and may guide the search for the development of additional new anticancer therapies.

Effect of surface treatment of ethylene vinyl acetate on the delamination of custom-fitted mouthguards.

BACKGROUND/AIM: Contamination of ethylene vinyl acetate (EVA) during mouthguard fabrication can cause delamination. The study evaluated the effects of different EVA surface treatments on the contact angle, laminate bond strength, and elongation capacity. MATERIALS AND METHODS: Specimens of two bonded EVA plates were prepared (n = 30). The Shore A hardness of standardized EVA plate specimens was measured before and after thermo-plasticization. The EVA plates were randomly allocated to one of five different surface treatment groups: no treatment (control); isopropyl alcohol, 100%; chloroform, 99.8%; self-cure acrylic resin monomer (methacrylate, ethylene glycol dimethacrylate, and chemical initiator-amine type); and ethyl alcohol, 70%. The maximum breaking force and elongation at the site of fracture were recorded using a universal testing machine. The contact angle surface was measured using ImageJ software. Scanning electron microscopy of the EVA surface was performed. The laminate bond strength was obtained by dividing the maximum breaking force by the bonding area between the two EVA plates. The laminate bond strength and maximum elongation data were analyzed by one-way ANOVA, followed by the Tukey's and the Dunnet test. The failure mode data was analyzed using the chi-square test (α = .05). RESULTS: EVA surface treatment significantly influenced the laminate bond strength and maximum elongation (p < .001). The control group had a higher contact angle and significantly lower laminate bond strength and maximum elongation than the other groups (p < .001). The acrylic resin monomer and chloroform-treated specimens had similar laminate bond strength and maximum elongation. The acrylic resin monomer group had a significantly lower contact angle (p < .001). CONCLUSIONS: All treatments had a significantly higher laminate bond strength and maximum elongation than the control group. The acrylic resin monomer and chloroform groups had a significantly higher laminate bond strength and maximum elongation and the acrylic resin monomer group had a lower contact angle than the other groups. The chloroform should be avoided due its hazardous effects.

Quality analysis of genomic DNA and authentication of fisheries products based on distinct methods of DNA extraction.

Molecular genetic techniques are an effective monitoring tool, but high-quality DNA samples are usually required. In this study, we compared three different protocols of DNA extraction: NaCl (saline); phenol-chloroform and commercial kit (Promega)-from three biological tissues of five individuals of Lutjanus purpureus under two methods of storage. The evaluated items included DNA concentration and purity, processing time and cost, as well as the obtaining of functional sequences. The highest average values of DNA concentration were obtained using the saline procedure and the commercial kit. Pure DNA was only obtained using the saline protocol, evaluated by the ratio of 260/280. The saline and phenol-chloroform protocols were the least expensive methods. The commercial kit costs are counterbalanced by the short time required. The procedure based on phenol-chloroform presented the worst results regarding DNA yield and the time required to perform all steps. The saline and commercial kit protocols showed similar results concerning the amount and quality of extracted DNA. Therefore, the final choice should be based on the available financial resources and the available time for carrying out each procedure of DNA extraction.

Exploration of non-phytopathogenic Aspergillus spp. isolates recovered from soil and compost as potential source of bioactive metabolites for potato Fusarium dry rot control.

During storage, infected potato tubers by Fusarium species leads to significant losses. Searching natural-based alternatives to chemical fungicides for the control of tuber dry rot pathogens is becoming essential. Nine Aspergillus spp. (A. niger, A. terreus, A. flavus, and Aspergillus sp.) isolates, recovered from soil and compost samples, were explored and evaluated for their ability to suppress Fusarium sambucinum the main causal agent of potato tuber dry rot disease in Tunisia. All conidial suspensions of Aspergillus spp. tested and their cell-free culture filtrates had significantly inhibited the in vitro pathogen growth by 18.5 to 35.9% and by 9 to 69% compared to control, respectively. A. niger CH12 cell-free filtrate was the most active against F. sambucinum at the three concentration tested (10, 15, and 20% v v-1). Chloroform and ethyl acetate extracts from four Aspergillus spp., tested at 5% v v-1, had limited F. sambucinum mycelial growth by 34-60% and 38-66%, respectively, compared to control, with A. niger CH12 ethyl extract being the most active. Tested on potato tubers inoculated with F. sambucinum, all tested Aspergillus spp. isolates, their cell-free filtrates and organic extracts had significantly decreased the external diameter of dry rot lesion compared to pathogen-inoculated and untreated control tubers. For the rot penetration, all Aspergillus spp. isolates, their organic extracts and only filtrates from A. niger CH12 and MC2 isolates had significantly limited dry rot severity compared to pathogen-inoculated and untreated control. The highest reductions in the external diameter of dry rot lesion (76.6 and 64.1%) and the average rot penetration (77.1 and 65.1%) were achieved using chloroform and ethyl acetate extracts from A. niger CH12, respectively. These results clearly demonstrated the presence of bioactive compounds in Aspergillus spp. that can be extracted and explored as an eco-friendly alternative for the control of the target pathogen.

Challenges in estimating the encapsulation efficiency of proteins in polymersomes - Which is the best method?

Abstract Polymersomes are nanometric vesicles that can encapsulate large and hydrophilic biomolecules, such as proteins, in the aqueous core. Data in literature show large variation in encapsulation efficiency (%EE) values depending on the method used for calculation. We investigated different approaches (direct and indirect) to quantify the %EE of different proteins (catalase, bovine serum albumin-BSA, L-asparaginase and lysozyme) in Pluronic L-121 polymersomes. Direct methods allow quantification of the actual payload of the polymersomes and indirect methods are based on the quantification of the remaining non-encapsulated protein. The protein-loaded polymersomes produced presented approximately 152 nm of diameter (PDI ~ 0.4). Higher %EE values were obtained with the indirect method (up to 25%), attributed to partial entanglement of free protein in the polymersomes poly(Ethylene Glycol) corona. For the direct methods, vesicles were disrupted with chloroform or proteins precipitated with solvents. Reasonable agreement was found between the two protocols, with values up to 8%, 6%, 17.6% and 0.9% for catalase, BSA, L-asparaginase and lysozyme, respectively. We believe direct determination is the best alternative to quantify the %EE and the combination of both protocols would make results more reliable. Finally, no clear correlation was observed between protein size and encapsulation efficiency.

Phytochemical Analysis and hypoglycemic potential of Filago hurdwarica (Wall. ex DC.) Wagenitz in alloxan induced diabetic mice.

Plants have profound therapeutic benefits, more economical treatments, fewer side effects, and a relatively cheap cost, making them a source of drugs for protective, preventative, curative, or conducive purposes and creating novel phytomedicines. Plant derived medicines are relatively safe compared to synthetic medicines. Many plants have proved to successfully aid in the treatment of diabetes including Filago hurdwarica (Wall. ex DC.) Wagenitz. The current investigations were therefore designed to assess the phytochemical, antioxidant, antidiabetic, and antihyperlipidemic activities of F. hurdwarica. The phytochemical investigations and antioxidant activities of different extracts were carried out using standard chemical tests, DPPH, and H2O2 scavenging assays. F. hurdwarica plant extract in Hydromethanolic solution were prepared by Soxhletation method and stored in refrigerator at 4°C for two days before use. Swiss Albino mice were made diabetic by a single dose of alloxan (150 mg/kg). Hydromethanolic plant extract and fractions of F. hurdwarica were screened for antidiabetic activity and given to the alloxan-induced diabetic mice at a concentration of 150-250 mg/kg of body weight in different groups of 6 diabetic mice each orally once a day for 15 days. Glibenclamide is also given to another group to as a standard drug to support the result at a dose of 10 mg/kg of body weight orally once a day for 15 days. Blood glucose levels and body weights of mice were measured on 0, 4, 7, 11 and 15th days. The study found that the extract was safe up to the dose level of 2000 mg/kg and the dose response effect of chloroform extract (150-250 mg/kg) of F. hurdwarica showed expressive antihyperglycemic effects and also improved other altered biochemical parameters associated with diabetes. The FTIR and XRD spectra demonstrated the occurrence of phenols, alcohols, alkenes, alkyl halides, ketones, and aromatic compounds and confirmed the amorphous nature of the extract. GC-MS spectral analysis showed the tentative presence of 31 phytochemical constituents in the chloroform extract of F. hurdwarica with different retention time. To conclude, the chloroform extract (250 mg/kg) of F. hurdwarica revealed considerable antioxidant, antihyperglycemic, and antihyperlipidemic potential and is safe for treating diabetes and related complications.

An investigation of the predominant structure of antibiotic azithromycin in chloroform solution through NMR and thermodynamic analysis.

Azithromycin (AZM) is a well-known macrolide-type antibiotic that has been used in the treatment of infections and inflammations. Knowledge of the predominant molecular structure in solution is a prerequisite for an understanding of the interactions of the drug in biological media. Experimental structural determination can be carried out for samples in solid-state (X-ray diffraction technique) and gas phase (electron diffraction experiment). In solution, spectroscopic methods can be used to extract valuable information which combined with quantum chemical calculations can lead to the determination of the preferred molecular structures to be observed when a given solute is dissolved in each solvent. That is precisely the aim of this work. We used experimental NMR chemical shift data (in CDCl3) as a reference for comparison with Density Functional Theory (DFT) NMR calculations, with geometry optimized having as guess input two crystallographic structures available in the literature with the configuration of all chiral carbon atoms inverted, named here A and B. The Polarizable Continuum Model (PCM) was used to describe the solvent effects (chloroform) including five explicit CHCl3 solvent molecules, which we believe can account for short and long-range solute-solvent interactions. Analysis of calculated thermodynamic, NMR chemical shift, MAE (Mean Absolute Error), and spin-spin coupling constant values revealed that both supposable C3R-C5S (named M2-A) and C3S-C5R (named M2-B) structures are equally probable to exist in chloroform solution. In addition, we found that the heavy atoms' conformation is reasonably similar in the solid-state and chloroform solution; however, regarding the OH groups, the spatial orientations are rather different with intramolecular OH⋯N and OH⋯O hydrogen bonds present in solution and with some of them being absent in the X-ray structure probably due to crystal packing effects.

Phytochemical Evaluation and Anti-Inflammatory Potential of Miconia albicans (Sw.) Triana Extracts.

The plant Miconia albicans (Sw.) Triana has been popularly used in Brazil to treat chronic inflammatory disturbances, such as osteoarthritis. This disease affects 250 million people worldwide, and is associated with intense pain and loss of articular function. There is a lack of information about the phytochemistry and bioactivity of M. albicans. Therefore, this study determined the chemical composition of some extracts and evaluated their cytotoxicity, along with their antioxidant and anti-inflammatory, activities using in vitro models. Aqueous and ethanolic extracts were prepared. Afterwards, a liquid-liquid partition was developed using chloroform, ethyl acetate, and n-butanol. The extracts were characterized by LC-MS, and their biological activities were evaluated on epithelial cells (Vero), tumoral hepatic cells (Hep-G2), and THP-1 macrophages. LC-MS analyses identified several flavonoids in all fractions, such as quercetin, myricetin, and their glycosides. The crude extracts and n-butanol fractions did not present cytotoxicity to the cells. The non-toxic fractions presented significant antioxidant activity when evaluated in terms of DPPH scavenging activity, lipid peroxidation, and ROS inhibition. THP-1 macrophages treated with the n-butanol fraction (250 µg/mL) released fewer pro-inflammatory cytokines, even in the presence of LPS. In the future, it will be necessary to identify the phytochemicals that are responsible for anti-inflammatory effects for the discovery of new drugs. In vivo studies on M. albicans extracts are still required to confirm their possible mechanisms of action.

EFFECT OF DIFFERENT SOLVENTS ON THE OPTICALLY STIMULATED LUMINESCENCE SIGNAL FROM MGB4O7:CE,LI-LOADED POLYMER FILMS.

The detailed dose analysis at the extremities remains a challenge, without affecting operators' mobility and their tactile sense. Using films loaded with optically stimulated luminescence (OSL) crystals have been studied in order to overcome some of these challenges in 2D dosimetry. In this work, we investigated flexible polymeric films loaded with MgB4O7:Ce,Li to acquire a better understanding of the dependence of the dosimetric signal characteristics on the production process and the influence of using different powder grain sizes. In film production, five different solvents were used: acetone-benzene, dichloromethane, chloroform, tetrahydrofuran and formic acid. Our results indicate that acetone-benzene is the solvent mixture that less influences the signal emitted by treated crystals, in comparison with the signal emitted by the pristine crystal powder. Conversely, by using formic acid, the crystalline structure of the sample was most severely modified, leading to a drastic reduction of the emitted OSL signal. We found that the extent of the grain surface in contact with the solvent in the process is important and should be taken into consideration when choosing the proper grain size to be used. Highlights  Polymeric films loaded with MgB4O7:Ce,Li crystals were produced using different solvents.Different effect on the OSL signal was found depending on the used solvent.Among the evaluated solvents, acetone-benzene was the one that less affected the OSL signal.

CARNOY'S SOLUTION INCREASES LYMPH NODES COUNT IN COLON CANCER SPECIMENS WHEN COMPARED TO FORMALIN FIXATION: A RANDOMIZED TRIAL.

AIM: At least 12 lymph nodes (LNs) should be examined following surgical resection of colon cancer. As it is difficult to find small LNs, fat clearing fixatives have been proposed, but there is no consensus about the best option. The objective of this study was to verify if Carnoy's solution (CS) increases the LN count in left colon cancer specimens. METHODS: A prospective randomized trial (clinicaltrials.gov registration: NCT02629315) with 60 patients with left colon adenocarcinoma who underwent rectosigmoidectomy. Specimens were randomized for fixation with CS or 10% neutral buffered formalin (NBF). After dissection, the pericolic fat from the NBF group was immersed in CS and re-dissected (Revision). The primary endpoint was the total number of LNs retrieved. RESULTS: Mean LN count was 36.6 and 26.8 for CS and NBF groups, respectively (p=0.004). The number of cases with <12 LNs was 0 (CS) and 3 (NBF, p=0.237). The duration of dissection was similar. LNs were retrieved in all cases during the revision (mean: 19, range: 4-37), accounting for nearly 40% of the LNs of this arm of the study. After the revision, no case was found in the NBF arm with <12 LNs. Two patients had metastatic LNs during the revision (no upstaging occurred). CONCLUSION: Compared to NBF, CS increases LN count in colon cancer specimens. After conventional pathologic analysis, fixing the pericolic fat with CS and performing a second dissection substantially increased the number of LNs.

Immunomodulatory effects of Blaps rynchopetera extract.

PURPOSE: To explore the potential immunomodulatory effects of total extract and different polar parts from Blaps rynchopetera Fairmaire. METHODS: Phagocytic activity was evaluated by neutral red assay, and the effect of the immune function was investigated by normal and immunocompromised mice models. RESULTS: In vitro, total extract, as well as chloroform, ethyl acetate, n-butanol and water fractions could individually enhance the phagocytic ability of mouse peritoneal macrophages; in addition, chloroform and ethyl acetate fractions had an increasing tendency when combined stimulation with lipopolysaccharide (LPS). In vivo, ethyl acetate fraction (EAF) could enhance the immune organ index, increase the serum hemolysin level and peripheral blood immune cells of immunocompromised mice, while for normal mice, the effect was inconspicuous. CONCLUSIONS: Blaps rynchopetera extracts had noteworthy immunomodulatory effect, especially for individuals with immune disorders.

Robust comparative performance of genomic DNA extraction methods from non-engorged phlebotomine sandflies.

The present study is a comparative analysis of DNeasy Blood & Tissue Qiagen® kit (Qiagen®, Hilden, Alemanha), salting out, HotShot and phenol-chloroform protocols to extract DNA from sandflies. In addition, a comparative test using sandflies with and without eyes evaluated the potential inhibitory effect in the cPCR. An inhibition test was performed using an exogenous DNA added to the qPCR. The genomic DNA quality of each sample was evaluated by cPCR based on the cytochrome c oxidase subunit I (cox1) gene. The DNA extraction protocols showed the following percentage of amplification: HotShot (91.6% [55/60]), salting out (71.6% [43/60]), phenol-chloroform (95% [57/60]) and kit DNeasy Blood & Tissue Qiagen® (73.3% [44/60]). The phenol-chloroform method achieved a significantly higher frequency of cox1 gene amplification. The pigment present in the phlebotomine's eyes seems to inhibit cPCR reactions since the frequency of amplification of the cox1 gene increased in the sandflies without eyes (p < 0.0001). The HotShot method showed the highest inhibitory potential. These manual extraction techniques can be an inexpensive and effective alternative to study vector-pathogen interactions.

Acute toxicity of disinfection by-products from chlorination of algal organic matter to the cladocerans Ceriodaphnia silvestrii and Daphnia similis: influence of bromide and quenching agent.

Algal organic matter (AOM) in water reservoirs is a worldwide concern for drinking water treatment; once it is one of the main precursors for disinfection by-products formation (DBPs). In this context, this study investigated the ecotoxicity of DBPs from chlorination of AOM to Ceriodaphnia silvestrii and Daphnia similis (Crustacea, Cladocera). The bioassays evaluated three scenarios, including the AOM extracted from Chlorella sorokiniana, the quenching condition used in the tests, and the DBPs formed after the chlorination of the two test waters with AOM (with and without bromide presence). The results showed that AOM has no toxic effects for the tested species under typical environmental concentration (5 mg∙L-1). However, since AOM is a potential precursor of DBPs, the toxicity of two test waters (TW-1 and TW-2) after the chlorination process (25 mg Cl2·L-1, for 7 days, at 20 °C) was tested. The sample with higher toxicity to the tested species was TW-1, in which chloroform and chloral hydrate were quantified (615 and 267 µg∙L-1, respectively). However, TW-2 showed lower concentration of chloroform and chloral hydrate (260 and 157 µg∙L-1, respectively), although bromodichloromethane, dibromochloromethane, and bromoform were also detected (464, 366, and 141 µg∙L-1, respectively). Although free chlorine is highly toxic to the tested species, the quenching conditions also affected the organisms' survival due to the use of ascorbic acid and the presence of reaction intermediates. Nonetheless, both species were more affected by TW-1 and TW-2 than the quenching condition. These results endorse the importance of removing the AOM before the disinfection process to avoid the formation of DBPs. In addition, ecotoxicological analyses could provide a more comprehensive assessment of water quality, especially considering the challenges of quantifying DBPs and other emerging contaminants.

Estudio de la actividad antimicrobiana de Xenophyllum poposum sobre microorganismos de la cavidad bucal por el método de difusión radial / Study of the antimicrobial activity of Xenophyllum poposum on microorganisms of the oral cavity by the radial diffusion method

Resumen En este estudio se evaluó la actividad antimicrobiana in vitro de extractos de Xenophyllum poposum sobre microorganismos bucales como Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, Actinomyces naeslundii, Actinomyces odontolyticus, Candida albicans y Veillonella sp. Se empleó el método de difusión radial en agar y como controles negativo y positivo de inhibición se emplearon etanol y clorhexidina al 0,12% (Plac out NF®) respectivamente. Los extractos con mayor actividad antimicrobiana fueron el etanólico y el clorofórmico. La diferencia entre ambos no fue estadísticamente significativa (p≥0,05). Tampoco se observó diferencia significativa con respecto a la clorhexidina, excepto sobre Veillonella sp., ya que el extracto etanólico presentó halos de inhibición significativamente menores sobre este microorganismo. Esto es importante ya que Veillonella se considera indicador de salud en relación a la caries dental. En base a esto, el extracto etanólico de Xenophyllum poposum podría ser usado como control químico de la biopelícula dental.

Abstract In this study, the in vitro antimicrobial activity of Xenophyllum poposum extracts on oral microorganisms such as Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, Actinomyces naeslundii, Actinomyces odontolyticus, Candida albicans, Veillonella sp. was evaluated. The radial diffusion method in agar was used and 0.12% ethanol and chlorhexidine (Plac out NF®) were used as negative and positive inhibition controls, respectively. The extracts with the highest antimicrobial activity were the ethanolic and chloroform extracts. The difference between the two was not statistically significant (p≥0.05). No significant difference was observed with respect to chlorhexidine, except on Veillonella sp., since the ethanolic extract presented significantly lower inhibition halos on this microorganism. This is important as Veillonella is considered an indicator of health in relation to dental caries. Based on this, the ethanolic extract of Xenophyllum poposum could be used as chemical control of dental biofilm.

Resumo Neste estudo, a atividade antimicrobiana de extratos de Xenophyllum poposum sobre microrganismos orais como Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, Actinomyces naeslundii, Actinomyces odontolyticus, Candida albicans e Veillonella sp. Foi utilizado o método de difusão radial em ágar e etanol 0,12% e clorexidina (Plac out NF®) como controles de inibição negativa e positiva, respectivamente. Os extratos com maior atividade antimicrobiana foram os extratos etanólico e clorofórmio. A diferença entre os dois não foi estatisticamente significativa (p≥0,05). Não foi observada diferença significativa em relação à clorexidina 0,12%, exceto em Veillonella sp., uma vez que o extrato etanólico apresentou halos de inibição significativamente menores neste microrganismo. Isso é importante, pois a Veillonella é considerada um indicador de saúde em relação à cárie dentária. Com base nisso, o extrato etanólico de Xenophyllum poposum pode ser utilizado como controle químico do biofilme dental.

Induction of apoptosis in A431 cells via ROS generation and p53-mediated pathway by chloroform fraction of Argemone mexicana (Pepaveraceae).

Argemone mexicana(Pepaveraceae) is an important medicinal plant commonly known as 'maxican prickly poppy' and is traditionally used to treat skin diseases. In the present study, the extract/fractions of aerial parts of A. mexicana after carrying out the organoleptic characteristics were sequentially extracted with the solvents of increasing polarities. Total fractions were examined for their radical scavenging activities in DPPH and DNA nicking assays. Among all, maximum antioxidant activity was shown by chloroform fraction (AmC) in DPPH assay with IC50 of 26.12 µg/ml, and DNA nicking assay showed 80.91% protective potential. The AmC fraction was analyzed for its antibacterial, cytotoxic potential, cell cycle analysis, mitochondrial membrane potential (MMP) and accumulation of reactive oxygen species (ROS) using A431 cell line. The AmC fraction exhibited remarkable antibacterial activity against bacterial strains in the order Klebsiella pneumoniae> Bacillussubtilis> Salmonella typhi> Staphylococcus epidermidis. The cytotoxic potential of the AmC fraction was analyzed in skin epidermoid carcinoma (A431) cells, osteosarcoma (MG-63) and cervical (HeLa) cell lines with a GI50 value of 47.04 µg/ml, 91.46 µg/ml and 102.90 µg/ml, respectively. The AmC fraction was extended further to explore its role in cell death using A431 cell line. Phase contrast and scanning electron microscopic studies on A431 cells exhibited all the characteristics indicative of apoptosis, viz., viability loss, cell shrinkage, cell rounding-off, DNA fragmentation and formation of apoptotic bodies. Flow cytometric analysis revealed enhanced ROS level, decreased MMP and arrest cell cycle at the G0/G1 phase further strengthened cell death by apoptosis. Increased expressions of apoptotic markers (p53, PUMA, cyt c, Fas and Apaf-1) were confirmed by RT-qPCR analysis. Furthermore, the AmC fraction was subjected to ultra-high-performance liquid chromatography, which revealed the presence of different polyphenols in the order: caffeic acid> epicatechin> kaempferol> chlorogenic acid> gallic acid> catechin> ellagic acid >umbeliferone> quercetin> coumaric acid. A critical analysis of results revealed that the AmC fraction induced cell death in epidermoid carcinoma cells via ROS and p53-mediated apoptotic pathway which may be ascribed to the presence of polyphenols in it.

Antidiabetic properties of oral treatment of hexane and chloroform fractions of Morus nigra leaves in streptozotocin-induced rats.

Morus nigra L. has been widely used in Brazilian folk medicine for the treatment of diabetes. We evaluate the chemical composition and antidiabetic properties of the hexane (Hex-Mn) and chloroform (Chlo-Mn) fractions obtained by partition of the crude ethanolic extract from the leaves in rats. Chemical composition analysis of Hex-Mn and Chlor-Mn was performed by gas chromatography-mass spectrometry (CG-MS). In vivo and in vitro studies were carried out to compare the antidiabetic activities of the Hex-Mn and Chlor-Mn fractions. Most of the compounds identified in Hex-Mn were α-linolenic acid, stigmast-5-en-3-ol and linolenic acid ethyl ester, while in Chlor-Mn, stigmast-5-en-3-ol, palmitic acid and α-linolenic acid were mainly identified. Only Hex-Mn treatment reduced both fasting and postprandial hyperglycemia. Additionally, Hex-Mn preserved body weight gain, preserved the hepatic glycogen content, and also reduced the thiobarbituric acid reactive substances and nitrite levels, as well as restored the superoxide dismutase. Furthermore, digestion of complex carbohydrates and intestinal glucose absorption was prevented by Hex-Mn treatment. Our results suggest that the antidiabetic activity of Hex-Mn may be explained, at least in part, by the insulin sensitivity increase, antioxidant properties and reduction in carbohydrate absorption in the small intestine.