Lupeol-induced nitric oxide elicits apoptosis-like death within Escherichia coli in a DNA fragmentation-independent manner.

Lupeol is known to be plentiful in fruits or plant barks and has an antimicrobial effect, however, its mode of action(s) has yet to be determined. To elucidate lupeol generates nitric oxide (NO), which is recognized for possessing an antimicrobial activity, intracellular NO was measured in Escherichia coli using DAF-FM. Using the properties of NO passing through plasma membrane easily, increased malondialdehyde levels have shown that lupeol causes lipid peroxidation, and the resulting membrane depolarization was confirmed by DiBAC4(3). These data indicated that lupeol-induced NO is related to the destruction of bacterial membrane. Further study was performed to examine whether NO, known as a cell proliferation inhibitor, affects bacterial cell division. As a result, DAPI staining verified that lupeol promotes cell division arrest, and followed by early apoptosis is observed in Annexin V/PI double staining. Even though these apoptotic hallmarks appeared, the endonuclease failed to perform properly with supporting data of decreased intracellular Mg2+ and Ca2+ levels without DNA fragmentation, which is confirmed using a TUNEL assay. These findings indicated that lupeol-induced NO occurs DNA fragmentation-independent bacterial apoptosis-like death (ALD). Additionally, lupeol triggers DNA filamentation and morphological changes in response to DNA repair system called SOS system. In accordance with the fact that ALD deems to SOS response, and that the RecA is considered as a caspase-like protein, increase in caspase-like protein activation occurred in E. coli wild-type, and no ΔRecA mutant. In conclusion, these results demonstrated that the antibacterial mode of action(s) of lupeol is an ALD while generating NO.

Endophytic Fungi from Dalbergia odorifera T. Chen Producing Naringenin Inhibit the Growth of Staphylococcus aureus by Interfering with Cell Membrane, DNA, and Protein.

This study focused on the antibacterial effects of the endophytic fungi producing naringenin from Dalbergia odorifera T. Chen against Staphylococcus aureus. The antibacterial activity was measured by the inhibition diameters, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The time-killing curve was also used to evaluate its antibacterial efficacy. The results of antibacterial activity determinations showed that endophytic fungi secondary metabolites can inhibit the growth of five pathogenic bacteria (S. aureus, Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, and Bacillus subtilis) and the most sensitive strain was S. aureus that had the MIC and MBC values of 0.13 and 0.50 mg/mL, respectively. The membrane permeability study was measured by a DNA leakage assay and electrical conductivity assay. Furthermore, the whole-cell protein lysates and DNA fragmentation assay was evaluated. The morphology of S. aureus treated with the endophytic fungi products was observed by scanning electron microscopy (SEM). The probable antibacterial mechanism of endophytic fungi secondary metabolites was the increased membrane permeability that leads to leaks of nucleic acids and proteins. SEM results further confirmed that the extracts can interfere with the integrity of S. aureus cell membrane and further inhibit the growth of bacteria, resulting in the death of bacteria. This study provides a new perspective for the antibacterial functions of endophytic fungi secondary metabolites for biomedical applications.

Effects of enrofloxacin treatment on the bacterial microbiota of milk from goats with persistent mastitis.

Antibiotic resistance has become a major concern for human and animal health. As fluoroquinolones have been extensively used in human and veterinary medicine, there has also been the rapid emergence and spread of antimicrobial resistance around the world. Here, we analysed the microbiome of goat milk using samples from healthy goats and those diagnosed with persistent mastitis and treated using the antibiotic enrofloxacin with 16S rRNA amplicon sequencing. We selected a group of 11 goats and 22 samples of milk that did not respond clinically to enrofloxacin treatment. Milk samples were evaluated before and after treatment to verify changes of the microbiota; the three first lactating goats were selected from the healthy control group. The milk samples from the healthy control animals presented a larger abundance of different species of bacteria of the Staphylococcus genus, but a smaller number of different genera, which indicated a more specific niche of resident bacteria. The Firmicutes phylum was predominantly different between the studied groups. Samples from before-treatment animals had a higher number of new species than those from the control group, and after being treated again. These microbiota received new bacteria, increasing the differences in bacteria even more in relation to the control group. Genotypes such as Trueperella and Mannheimia, between other genera, had a high abundance in the samples from animals with persistent mastitis. The dysbiosis in this study, with marked evidence of a complex microbiota in activity in cases of the failure of antimicrobial treatment for persistent chronic mastitis, demonstrates a need to improve the accuracy of pathogen identification and increases concern regarding antibiotic treatments in milk production herds.

Biological Activities and Secondary Metabolite Screening of Rumex hastatus Extract through Fourier Transform Infrared and Raman Spectroscopy.

BACKGROUND: Human infectious diseases are caused by various pathogens including bacteria, fungi, viruses, parasites, and protozoans. These infectious agents are controlled by using synthetic drugs as well as natural sources. OBJECTIVE: The aim of current study was to evaluate the antibacterial effect of Rumex hastatus against clinical bacterial pathogens. METHODS: In current research antibacterial effect of Rumex hastatus was analyzed against seven clinical pathogenic bacteria such as Escherichia coli, Serratia marcescens, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa through agar well diffusion method. The boiled extract was used for the phytochemical screening, antioxidant potential, thin layer chromatography, bio-autography, and spot screening. Genomic DNA was extracted to find the DNA protection effect of R. hastatus. RESULTS: Antibacterial results showed that diethyl ether extract has the maximum inhibition of S. pyogenes (9.66 ± 0.57 mm). Acetone and diethyl ether extracts showed moderate inhibition of K. pneumoniae (6.33 ± 1.52 mm and 5.66 ±1.15 mm) and S. aureus (6.33 ± 1.52 mm and 5.66 ± 0.57 mm). Similarly, chloroform extract indicated moderate inhibition of S. pyogenes (5.66 ± 1.15 mm). Ethanol extract had low or even no effect on the growth of bacteria. Genomic DNA extraction also encouraged the antibacterial effect of R. hastatus. Various phytochemical constituents such as ketoses, oligosaccharides, amino acids, amines, sugars, flavonoids, and antioxidant constituents were detected. TLC-Bioautography and spot screening results revealed the potential use of R. hustatus as an antibacterial agent. CONCLUSION: It was concluded that most of the tested fractions appeared as an important source for the discovery of new antimicrobial drugs.

Genotoxic and cytotoxic potential of Alternanthera Bettzickiana, an important ethno-medicinal plant.

The present study was carried out to investigate the mutagenic and cytotoxic potential of n-hexane and aqueous-methanolic whole plant extracts of Alternanthera bettzickiana. Aqueous-methanolic and n-hexane extracts of Alternanthera bettzickiana extracts were assessed for the mutagenic potential with Salmonella tester strains TA-100 and TA-102 in the presence and absence of the rodent enzyme activation system and cytotoxic potential was assessed by MTT assay. Aqueous-methanolic extract showed the presence of saponins, tannins, terpenoids, flavonoids and glycosides. However n-hexane extract revealed the presence of tannins and terpenoids only. It was found that a concentration as low as 15mg/mL of both extracts was more mutagenic to the TA 102 tester strain than TA-100. Hexane whole plant extract of Altenanthera bettzickiana was more mutagenic than aqueous-methanolic extract considering revertant colonies of TA 100 strain. Aqueous-methanolic and n-hexane whole plant extracts of Altenanthera bettzickiana showed higher mutagenic potential in the presence of the enzyme activation system. Mutagenicity of aqueous-methanolic extract increased with an enzyme activation system in case of TA 100 whereas mutagenicity of n-hexane extract decreased in the presence of the enzyme activation system with TA 100 and TA 102 strains. Aqueous-methanolic and n-Hexane whole plant extracts of Alternanthera bettzickiana showed an IC-50 of 493 and 456 µg/mL in BHK-21 cells respectively. It can be concluded that Altenanthera bettzickiana exhibited mutagenic activity in a bacterial reverse mutation assay with and without enzyme activation systems. However, it showed limited cytotoxicity to BHK-21 cells.

Effect of secondary metabolite of Actinidia deliciosa on the biofilm and extra-cellular matrix components of Acinetobacter baumannii.

Acinetobacter baumannii, opportunistic nosocomial pathogen, increases gradually in the clinical setup. The high level of resistance mechanisms acquired by these bacteria makes their eradication difficult and biofilm formation is one of them. Biofilm comprises of closely packed bacterial population crowded together by extra-cellular matrix (ECM). ECM contains bacterial secreted polymers such as exopolysaccharides (EPS), proteins and extracellular-DNA (e-DNA) and rarely amyloidogenic proteins. Biofilm offers protection of underlying bacterial population against chemotherapeutic agents and host immune system. Therefore, present efforts are focused to find a novel therapeutic that targets biofilm-associated infections. Plants are used as a natural therapeutic for numerous ailments. In order to find an alternative of the available antibacterial drugs, we have focused on the natural herbal active compounds. In this study, we have extracted active compounds from various medicinal plants and screened its anti-biofilm activity against carbapenem resistant strain of A. baumannii. Results showed that polar extract of kiwi (Actinidia deliciosa) and clove (Syzygium aromaticum) exhibit effective anti-biofilm activity. These two plants were also used for their phytochemical screening and TLC profiling to find out the constituting secondary metabolites. Actinidia deliciosa extract contains an alkaloid (sanquinarine) as well as a flavonoid (hydroxyflavone). Anti-biofilm effect of this extract on the ECM of A. baumannii showed that it reduces EPS, protein and eDNA contents in the ECM. Proteins of ECM have also shown to form amyloid like structure, which was evident from its interaction with the Congo Red. CFU counting after Actinidia deliciosa extract treatment also supported the results. Therefore, it can be concluded that polar extract of A. deliciosa can be used to find suitable alternative therapeutic to control biofilm formation by carbapenem resistant strain of Acinetobacter baumannii.

Antioxidant and antimutagenic potential of Psidium guajava leaf extracts.

Fruits, vegetables and medicinal herbs rich in phenolics antioxidants contribute toward reduced risk of age-related diseases and cancer. In this study, Psidium guajava leaf extract was fractionated in various organic solvents viz. petroleum ether, benzene, ethyl acetate, ethanl and methanol and tested for their antioxidant and antimutagenic properties. Methanolic fraction showed maximum antioxidant activity comparable to ascorbic acid and butylated hydroxyl toluene (BHT) as tested by DPPH free radical scavenging, phosphomolybdenum, FRAP (Fe3 + reducing power) and CUPRAC (cupric ions (Cu2+) reducing ability) assays. The fraction was analyzed for antimutagenic activities against sodium azide (NaN3), methylmethane sulfonate (MMS), 2-aminofluorene (2AF) and benzo(a)pyrene (BP) in Ames Salmonella tester strains. The methanol extracted fraction at 80 µg/ml concentration inhibited above 70% mutagenicity. Further, phytochemical analysis of methanol fraction that was found to be most active revealed the presence of nine major compounds by gas chromatography-mass spectrometry (GC-MS). This data suggests that guava contains high amount of phenolics responsible for broad-spectrum antimutagenic and antioxidant properties in vitro and could be potential candidates to be explored as modern phytomedicine.

The mutagenic, antimutagenic and antioxidant properties of Hypericum lydium.

CONTEXT: There is a growing market demand for Hypericum sp., a pharmacologically active plant that has been traditionally used to treat various ailments. However, there have been limited studies on the extract or essential oil of Hypericum lydium Boiss (Hypericaceae). OBJECTIVE: This study investigates for the first time the antioxidant, mutagenic and antimutagenic activity of an ethanol extract of H. lydium. MATERIAL AND METHODS: Ethanol extract from aerial parts of H. lydium harvested from Turkey were tested for this mutagenic and antimutagenic activities (2.0-0.002 mg/plate) using Ames Salmonella/microsome test system. 4-Nitro-o-phenylenediamine (4-NPD) (3 µg/plate) for the Salmonella typhimurium TA98 and sodium azide (NaN3) (8 µg/plate) for the S. typhimurium TA100 were used as positive controls. The antioxidant activity, total antioxidant activity and phenolic constituent of the extract (2.0-0.002 mg/mL) was determined by the inhibition of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), ß-carotene-linoleic acid model and by means of Folin-Ciocalteu reagent, respectively. RESULTS: The extract showed no sign of mutagenicity at the tested concentrations (0.002-2.0 mg/mL), and showed concentration-dependent antimutagenic activity against NaN3 and 4-NPD ranging from 26.8 to 81.5%. The extract was found to be an efficient scavenger of DPPH (IC50 0.165 ± 0.23 mg/mL) and to inhibit ß-carotene-linoleic acid bleaching (IC50 0.39 ± 0.11 mg/mL). DISCUSSION AND CONCLUSION: These findings indicate ethanol extract of H. lydium to be a safe and effective agent that may be incorporated into new strategies for the prevention of cancer and mutagenesis.

Safety assessment of a traditionally used extract from leaves of Boldoa purpurascens.

ETHNOPHARMACOLOGICAL RELEVANCE: Boldoa purpurascens Cav. (Nyctaginaceae) is a plant species used in traditional medicine in Cuba as a diuretic. AIM OF THE STUDY: The aim of the present investigation was to evaluate the safety profile of a hydroalcoholic extract from leaves of Boldoa purpurascens. MATERIALS AND METHODS: First, an experimental study to assess the oral acute toxicity at a dose of 2000mg/kg body weight of the extract was carried out. Potential genotoxicity of the extract was evaluated using the Ames test and the micronucleus induction assay in mouse bone marrow. In the Ames test a concentration range of 50, 100, 150, 300 and 500µg/plate was tested. In the micronucleus induction assay, doses of 500, 1000 and 2000mg/kg of body weight were tested. For completeness, since the extract contains saponins, the evaluation of the hemolytic activity, ocular and skin irritation were included. RESULTS: No signs or symptoms of toxicity were observed in the oral acute toxicity test (body weight at baseline, seven days and end of the experiment of 236.41±20.07, 256.81±30.44 and 240.02±26.16 respectively for the treated group). The hydroalcoholic extract from the leaves was not mutagenic in the Ames test, and no genotoxicity was observed in the micronucleus assay. A hemolysis test at concentration of 1mg/mL confirmed hemolytic activity, which is not a safety concern since saponins are not absorbed after oral administration. In order to evaluate the percentage of protein denaturation, the ocular irritability index was calculated. The extract was found to be irritating. Finally, skin irritability was evaluated and the irritation index was equal to zero. CONCLUSIONS: Based on the toxicological evaluation of a traditionally used hydroalcoholic extract from the leaves of Boldoa purpurascens we can confirm the safety of its oral use.

Design, Synthesis, and Antimicrobial Evaluation of Novel Quinolone Imidazoles and Interactions with MRSA DNA.

A novel series of quinolone imidazoles as new type of antimicrobial agents were synthesized. Most compounds exhibited good bioactivities especially against MRSA even superior to reference drugs. They induced bacterial resistance more slowly than clinical drugs and gave low cytotoxicity to human cells. The pKa values of these compounds showed appropriate ranges to pharmacokinetic behaviors. The interactions between compound 8b, Cu(2+) ion, and MRSA DNA revealed that compound 8b could intercalate into DNA through copper ion bridge to form a steady 8b-Cu(2+) -DNA ternary complex which might further block DNA replication to exert the powerful bioactivities. Study of compound 8b with human serum albumin indicated that compound 8b could be effectively stored and carried by human serum albumin.

Safety evaluation of standardized allergen extract of Japanese cedar pollen for sublingual immunotherapy.

Japanese cedar (JC) pollinosis is caused by Japanese cedar pollen (JCP) and most common seasonal allergic disease in Japan. Subcutaneous immunotherapy (SCIT) with allergen extract of JCP (JCP-allergen extract) is well established for JC pollinosis treatment with improvement of symptoms. However, major drawbacks for SCIT are repeated painful injections, frequent hospital visits and anaphylactic risk. Currently, sublingual immunotherapy (SLIT) has received much attention as an advanced alternative application with lower incidence of systemic reactions because the liquid or tablet form of allergen is placed under the tongue. The aim of this study was safety evaluation of standardized JCP-allergen extract currently developed for SLIT in JC pollinosis. JCP-allergen extract showed no potential genotoxicity. No systemic effects were observed in rats administered JCP-allergen extract orally for 26 weeks followed by 4-week recovery period. Mild local reactions such as hyperplasia and increased globule leukocytes resulting from vehicle (glycerin)-induced irritation were observed in stomach. No-observed-adverse-effect level was greater than 10,000 JAU/kg/day for systemic toxicity, equivalent to 300-fold the human dose. No local irritation was found in rabbits oral mucosae by 7-day sublingual administration. These results demonstrate the safe profile of standardized JCP-allergen extract, suggesting it is suitable for SLIT in JC pollinosis.

Genotoxicity studies on the root extract of Polygala tenuifolia Willdenow.

The root of Polygala tenuifolia Willdenow has been used for the treatment against insomnia, amnesia, depression, palpitations with anxiety, and memory improvement. However, there is no sufficient background information on toxicological evaluation of the root to given an assurance of safety for developing dietary supplements and functional foods. As part of a safety evaluation, the potential genotoxicity of the root extract of P. tenuifolia was evaluated using a standard battery of tests (bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay). In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, the extract did not increase the number of revertant colonies in any tester strain with or without metabolic activation by S9 mix, and did not cause chromosomal aberration in short-period test with the S9 mix or in the continuous (24h) test. A bone marrow micronucleus test in ICR mice dosed by oral gavage at doses up to 2000 mg/kg/day showed no significant or dose dependent increase in the frequency of micronucleated polychromatic erythrocytes (PCE). These results indicate that ingesting the rot extract P. tenuifolia is not genotoxic at the proper dose.

Synthesis, DNA cleavage and antimicrobial activity of 4-thiazolidinones-benzothiazole conjugates.

Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC's were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C. albicans with MIC values in the range of 15.6-125 microg/ml. Preliminary study of the structure-activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect on the antimicrobial activity of these compounds and contributes positively for the action. DNA cleavage experiments gave valuable hints with supporting evidence for describing the mechanism of action and hence showed a good correlation between their calculated MIC's and its lethality.

Genotoxic Maillard byproducts in current phytopharmaceutical preparations of Echinodorus grandiflorus.

Extracts of Echinodorus grandiflorus obtained from dried leaves by three different techniques were evaluated by bacterial lysogenic induction assay (Inductest) in relation to their genotoxic properties. Before being added to test cultures, extracts were sterilized either by steam sterilization or ultraviolet light. Only the extracts prepared by infusion and steam sterilized have shown genotoxic activity. The phytochemical analysis revealed the presence of the flavonoids isovitexin, isoorientin, swertisin and swertiajaponin, isolated from a genotoxic fraction. They were assayed separately and tested negative in the Inductest protocol. The development of browning color and sweet smell in extracts submitted to heat, prompted further chemical analysis in search for Maillard's reaction precursors. Several aminoacids and reducing sugars were cast in the extract. The presence of characteristic Maillard's melanoidins products was determined by spectrophotometry in the visible region and the inhibition of this reaction was observed when its characteristic inhibitor, sodium bisulfite, was added prior to heating. Remarkably, this is the first paper reporting on the appearance of such compounds in a phytomedicine preparation under a current phytopharmaceutical procedure. The genotoxic activity of such heat-prepared infusions imply in some risk of developing degenerative diseases for patients in long-term, uncontrolled use of such phytomedicines.

A novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli.

Silver nanoparticles are known to have antimicrobial properties and have been used extensively in medicine, although the mechanism(s) of action have not yet been clearly established. In the present study, the findings suggest a novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli, namely, the induction of a bacterial apoptosis-like response. We propose a possible mechanism for the bacterial apoptosis-like response that includes the following: accumulation of reactive oxygen species (ROS) (detected with H2DCFDA staining), increased intracellular calcium levels (detected with Fura-2 AM), phosphatidylserine exposure in the outer membrane (detected with Annexin V) which is the hallmarks of early apoptosis, disruption of the membrane potential [detected with DiBAC4(3)], activation of a bacterial caspase-like protein (detected by FITC-VAD-FMK staining) and DNA degradation (detected with TUNEL assay) which is the hallmarks of late apoptosis in bacterial cells treated with silver nanoparticles. We also performed RecA expression assay with western blotting and observed activation of SOS response to repair the damaged DNA. To summarize, silver nanoparticles are involved in the apoptosis-like response in E. coli and the novel mechanisms which were identified in this study, suggest that silver nanoparticles may be an effective antimicrobial agent with far lower propensity for inducing microbial resistance than antibiotics.

Development of a single multiplex amplification refractory mutation system PCR for the detection of rifampin-resistant Mycobacterium tuberculosis.

A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations.

[Evaluation of cytotoxic effect and genetic damage of standardized extracts of Solanum nudum with antiplasmodial activity]. / Evaluación del efecto citotóxico y del daño genético de extractos estandarizados de Solanum nudumcon actividad anti- Plasmodium.

INTRODUCTION: The plant Solanum nudum (Solanaceae) is extensively used for the treatment of malaria-related symptoms in traditional medicine practices in the Colombian Pacific. Recently, it has become a significant source of promising new molecules for developing a pharmaceutical malaria treatment. OBJECTIVE: This research aimed to evaluate the cytotoxic effect and the genetic damage of standardized extracts of S. nudumon different cells. MATERIALS AND METHODS: Sixty six standardized S. nudum extracts were used, evaluating cytotoxicity in U937 and HepG2 cells and the antiplasmodial activity using both a chloroquine-sensitive (NF54) and a chloroquine-resistant (FCB2) strain. The hemolytic effect on healthy O + erythrocytes, the mutagenic effect on S.Typhimurium TA98 and TA100 strains and the genotoxic effect on U937 cells were evaluated. The extracts that displayed both antiplasmodial activity and low toxicity were selected. RESULTS: Five extracts were selected: 28MA1, 29MA1, 51MA1, 55MA1 and 61MA1. These extracts were active against P. falciparum with IC 50 between 9.8 and 54.8 µg/ml and selectivity indexes were calculated between 0.9 and 4.4, the latter for 29MA1. Also, no hemolytic effects in healthy O + erythrocytes were shown at a concentration of 250 µg/ml, nor did they cause mutations in the TA98 and TA100 strains or generate genotoxic effects in U937cells. CONCLUSION: The use of standardized extracts of S. nudum could contribute to the body of work aimed at developing a new pharmaceutical treatment for malaria using natural products.

Preclinical toxicological evaluations of the sclerotium of Lignosus rhinocerus (Cooke), the Tiger Milk mushroom.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerus (Tiger Milk mushroom) is distributed in South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. In Malaysia, it is the most popular medicinal mushroom used by the indigenous communities to relieve fever, cough, asthma, cancer, food poisoning and as a general tonic. In China, this mushroom is an expensive traditional medicine used to treat liver cancer, chronic hepatitis and gastric ulcers. The sclerotium of the mushroom is the part with medicinal value. This rare mushroom has recently been successfully cultivated making it possible to be fully exploited for its medicinal and functional benefits. The present study was carried out to evaluate the chronic toxicity of the sclerotial powder of Lignosus rhinocerus cultivar (termed TM02), its anti-fertility and teratogenic effects as well as genotoxicity. MATERIALS AND METHODS: Sprague Dawley rats (10 rats/group/sex) were fed orally with 250, 500 and 1000 mg/kg of sclerotial powder of TM02. The sclerotial powder was orally administered once daily and consecutively for 180 days. At the completion of the oral feeding period, analysis of hematological and clinical biochemical parameters, urine profiles, organ weight as well as histopathological analysis were carried out. The effect of the sclerotial powder on fertility and its possible teratogenicity were examined by feeding rats orally with 100 mg/kg sclerotial powder consecutively for 7-8 weeks. Genotoxicity was evaluated by Ames test using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. RESULTS: The results showed that oral administration of the sclerotial powder of the Lignosus rhinocerus cultivar at daily dose of up to 1000 mg/kg for 180 days had no adverse effect on the general clinical observations, body weight, hematology, clinical biochemistry, urinalysis, absolute organ weight as well as relative organ weight, nor induced histological changes in the organs. Oral administration of 100 mg/kg sclerotial powder of the Lignosus rhinocerus for 7-8 weeks did not affect the fertility of the rats nor induce teratogenic effect on their offspring. Lignosus rhinocerus sclerotial powder up to 5000 µg/plate in the presence and absence of metabolic activation did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. CONCLUSION: Our results showed that the no-observed-adverse-effect level (NOAEL) dose of the sclerotial powder of Lignosus rhinocerus in 180-day chronic toxicity study is more than 1000 mg/kg. Oral feeding of the sclerotial powder at 100mg/kg did not induce adverse effect on rats' fertility nor causing teratogenic effect on their offspring. In the reverse mutation Ames test, the sclerotial powder at all tested concentration did not show any genotoxicity.

Evaluación del efecto citotóxico y del daño genético de extractos estandarizados de Solanum nudumcon actividad anti- Plasmodium / Evaluation of cytotoxic effect and genetic damage of standardized extracts of Solanum nudum with antiplasmodial activity

Introducción. La planta Solanum nudum es ampliamente usada en la medicina tradicional del Pacífico colombiano para tratar las fiebres y la malaria, o paludismo, y se ha convertido en una fuente de nuevas moléculas promisorias. Objetivo. Evaluar el efecto citotóxico y daño genético de extractos estandarizados de S. nudum en diferentes modelos celulares. Materiales y métodos. A 66 extractos estandarizados de S. nudum se les evaluó la actividad anti- Plasmodiumin vitro en dos cepas de Plasmodium falciparum, una sensible (NF54) y otra resistente (FCB2) a la cloroquina, y la citotoxicidad en células U937 y HepG2. Se seleccionaron los extractos que presentaron actividad anti- Plasmodium y baja toxicidad, y se les estimó su efecto hemolítico en eritrocitos sanos O + , el efecto mutagénico en las cepas TA98 y TA100 de Salmonella Typhimurium y el efecto genotóxico en células U937. Resultados. Se seleccionaron cinco extractos como promisorios (28MA1, 29MA1, 51MA1, 55MA1 y 61MA1), los cuales fueron activos en las cepas de P. falciparum con concentración inhibitoria 50 (CI 50 ) entre 9,8 y 54,8 µg/ml. El extracto 29MA1 fue el más selectivo para Plasmodium, con índice de selectividad de 4,4 y 14,5 para las células U937 y HepG2, respectivamente. En ningún extracto se observó efecto hemolítico a 250 µg/ml, no causaron mutaciones en las cepas TA98 y TA100 de S.Typhimurium, ni generaron efectos genotóxicos en células U937. Conclusiones. La utilización de extractos estandarizados de S. nudum contribuye con los trabajos encaminados al desarrollo de una nueva formulación farmacéutica para tratar la malaria a partir de productos naturales.

Introduction. The plant Solanum nudum (Solanaceae) is extensively used for the treatment of malaria-related symptoms in traditional medicine practices in the Colombian Pacific. Recently, it has become a significant source of promising new molecules for developing a pharmaceutical malaria treatment. Objective. This research aimed to evaluate the cytotoxic effect and the genetic damage of standardized extracts of S. nudumon different cells. Materials and methods. Sixty six standardized S. nudum extracts were used, evaluating cytotoxicity in U937 and HepG2 cells and the antiplasmodial activity using both a chloroquine-sensitive (NF54) and a chloroquine-resistant (FCB2) strain. The hemolytic effect on healthy O + erythrocytes, the mutagenic effect on S.Typhimurium TA98 and TA100 strains and the genotoxic effect on U937 cells were evaluated. The extracts that displayed both antiplasmodial activity and low toxicity were selected. Results. Five extracts were selected: 28MA1, 29MA1, 51MA1, 55MA1 and 61MA1. These extracts were active against P. falciparum with IC 50 between 9.8 and 54.8 µg/ml and selectivity indexes were calculated between 0.9 and 4.4, the latter for 29MA1. Also, no hemolytic effects in healthy O + erythrocytes were shown at a concentration of 250 µg/ml, nor did they cause mutations in the TA98 and TA100 strains or generate genotoxic effects in U937cells. Conclusion. The use of standardized extracts of S. nudum could contribute to the body of work aimed at developing a new pharmaceutical treatment for malaria using natural products.

Influence of dietary blueberry and broccoli on cecal microbiota activity and colon morphology in mdr1a(-/-) mice, a model of inflammatory bowel diseases.

OBJECTIVE: Enteric microbiota has been shown to be associated with various pathological conditions such as inflammatory bowel disease (IBD). This study aimed to determine the anti-inflammatory colonic effects of blueberries and broccoli in mdr1a(-/-) mice (IBD mouse model) through modification of microbiota composition in the gastrointestinal tract. METHODS: The mdr1a(-/-) mice were fed either a control diet or the control diet supplemented with either 10% blueberry or broccoli for 21 wk. We investigated the influence of these diets on cecal microbiota and organic acids, colon morphology, and bacterial translocation to mesenteric lymph nodes. RESULTS: In comparison to mice fed the control diet, blueberry and broccoli supplementation altered cecum microbiota similarly with the exception of Faecalibacterium prausnitzii, which was found to be significantly lower in broccoli-fed mice. High concentrations of butyric acid and low concentrations of succinic acid were observed in the cecum of broccoli-fed mice. Blueberry- and broccoli-supplemented diets increased colon crypt size and the number of goblet cells per crypt. Only the broccoli-supplemented diet significantly lowered colonic inflammation compared to mice fed the control diet. Translocation of total microbes to mesenteric lymph nodes was lower in broccoli-fed mice compared to blueberry and control diet groups. CONCLUSION: Dietary blueberries and/or broccoli altered the composition and metabolism of the cecal microbiota and colon morphology. Overall, these results warrant further investigation through clinical studies to establish whether the consumption of blueberries and/or broccoli is able to alter the composition and metabolism of large intestine microbiota and promote colon health in humans.