Antileishmanial activity and trypanothione reductase effects of terpenes from the Amazonian species Croton cajucara Benth (Euphorbiaceae)

BackgroundLeishmaniasis comprises several infectious diseases caused by protozoa parasites of Leishmania genus. In recent years, there has been a growing interest in the therapeutic use of natural products to treat parasitic diseases. Among them Croton cajucara Benth. (Euphorbiaceae) is a plant found in the Amazonian region with a history of safe use in folk medicine.PurposeThe purpose of this study was to investigate the effects of clerodane diterpenes, trans-dehydrocrotonin (DCTN), trans-crotonin (CTN) and acetylaleuritolic acid (AAA) obtained from powdered bark of C. cajucara against promastigotes, axenic and intracellular amastigotes of Leishmania amazonensis. Furthermore, the effects of DCTN and CTN on the trypanotiona reductase enzyme were also investigated. The extraction of the terpenes was carried out as previously reported (Maciel et al., 1998, Maciel et al., 2003).MethodsThe effect of the isolated compounds (DCTN, CTN and AAA) from the bark of C. cajucara was assessed in vitro against promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis by counting of remaining parasites in a Neubauer chamber in comparison to pentamidine used as standard drug. The action of natural products on trypanothione reductase was assessed using soluble protein fraction of promastigotes. The assays were performed by incubation with HEPES, EDTA, NADPH and trypanothione disulfide to quantify the NAPH consumption by TryR.ResultsThe results showed very high efficacy, especially of the diterpene DCTN, against promastigotes (IC₅₀ = 6.30 ± 0.06 µg/ml) and axenic amastigotes (IC₅₀ = 19.98 ± 0.05 µg/ml) of L. amazonenesis. The cytotoxic effect of the best active natural product was evaluated on mouse peritoneal infected macrophages (IC₅₀ = 0.47 ± 0.03 µg/ml in 24 h of culture), and the treatment revealed that DCTN never reaches toxic concentrations while reducing the infection and, most importantly, with no toxicity (>100 µg/ml with 0% of macrophage kill) when compared to pentamidine (37.5 µg/ml with 100% of macrophage kill). Furthermore, all of the natural products assayed on the trypanothione reductase enzyme inhibited the enzyme activity compared to the control.ConclusionClerodane diterpenes from C. cajucara showed promising in vitro antileishmanial effects against L. amazonensis, specially the DCTN with no macrophage toxicity up to the assayed concentration. In addition, the action on trypanothione reductase enzyme revealed a possible mechanism of action.     

Piper crocatum Ruiz & Pav. ameliorates wound healing through p53, E-cadherin and SOD1 pathways on wounded hyperglycemia fibroblasts

IntroductionPiper crocatum Ruiz & Pav (P. crocatum) has been reported to accelerate the diabetic wound healing process empirically. Some studies showed the benefits of P. crocatum in treating various diseases but its mechanisms in diabetic wound healing have never been reported. In the present study we investigated the diabetic wound healing activity of the active fraction of P. crocatum on wounded hyperglycemia fibroblasts (wHFs).MethodsBioassay-guided fractionation was performed to get the most active fraction. The selected active fraction was applied to wHFs within 72 h incubation. Mimicking a diabetic condition was done using basal glucose media containing an additional 17 mMol/L D-glucose. A wound was simulated via the scratch assay. The collagen deposition was measured using Picro-Sirius Red and wound closure was measured using scratch wound assay. Underlying mechanisms through p53, αSMA, SOD1 and E-cadherin were measured using western blotting.ResultsWe reported that F_IV is the most active fraction of P. crocatum. We confirmed that F_IV\(7.81 µg/ml, 15.62 µg/ml, 31.25 µg/ml, 62.5 µg/ml, and 125 µg/ml) induced the collagen deposition and wound closure of wHFs. Furthermore, F_IV treatment (7.81 µg/ml, 15.62 µg/ml, 31.25 µg/ml) down-regulated the protein expression level of p53 and up-regulated the protein expression levels of αSMA, E-cadherin, and SOD1.Discussion/conclusionsOur findings suggest that ameliorating collagen deposition and wound closure through protein regulation of p53, αSMA, E-cadherin, and SOD1 are some of the mechanisms by which F_IV of P. crocatum is involved in diabetic wound healing therapy.     

Total phenolic,flavonoid contents,and biological activities of stem extracts of Astragalus spinosus (Forssk.) Muschl. grown in Northern Border Province,Saudi Arabia

Background and objectiveGenus Astragalus belongs to the family Fabaceae and is one among the largest genera consisting of around 3000 species. The plants have been used traditionally in treatment of various ailments in folklore. The study was planned to assess the analgesic and inflammatory activity of Astragulus spinosus (Forssk.) Muschl extract of the stem.Materials and methodsCourse powder of stems of Astragulus spinosus was extracted using chloroform and methanol as solvents. Folin ciocalteu method was employed for determination of the phenolic acid content. Aluminum chloride colorimetric procedure was followed for estimating the flavonoid content. Both chloroform and methanolic extracts at 250 and 500 mg/kg, were tested for the analgesic activity, however, only methanolic extract was selected for anti-inflammatory property based on the results of analgesic activity. The analgesic effect was executed on male rats by the hot plate model. The anti-inflammatory effect was studied in the carrageenan rat paw edema model. The experimental information was interpreted statistically using one-way ANOVA and p < 0.05 was used to express importance of the results.ResultsThe total phenolics of the methanol extract was 420 µg and that of chloroform extract was 265 µgwhile total flavonoid content in terms of quercetin was found to be 68 µg and 17.5 µg for methanol and chloroform extract respectively. Only methanolic extract exhibited significant (p < 0.001) analgesic activity by elevating the pain threshold starting from 15 min. The methanolic extract inhibited (p < 0.001) the edema in carrageenan induced model. The performance of higher dose (500 mg/kg) was better with reference to lower dose (250 mg/kg).ConclusionOutcome of the results show that the methanolic stem extracts exhibited significant analgesic and anti-inflammatory-like activity with reference to chloroform extract. Credit of which is given to flavonoids and phenolic content present in the methanolic extract. However, more research is suggested to establish the safety and effectiveness of the herbal drug to manage diseased states including pain and inflammation.     

Antagonistic,Anti-oxidant,Anti-inflammatory and Anti-diabetic Probiotic Potential of Lactobacillus agilis Isolated From the Rhizosphere of the Medicinal Plants

Potential probiotic bacteria can be used as a biotherapeutic agent and a sustainable alternative to antibiotics, as an anti-oxidative, anti-inflammatory, and anti-diabetic agent without causing any serious side effects. Mostly human-friendly Lactic acid bacteria (LAB) have been isolated from the animal-human origin to be used as biotherapeutic agents or to produce useful metabolites (nutraceutical). However, less information is known about the role of medicinal plants associated LAB as biotherapeutic agents. The isolation of 115 human-friendly Lactobacillus strains was done from the rhizosphere of the medicinal plants Ocimum tenuiflorum, Azadirachta indica, Ficus carica. The obtained bacteria were then tested for their safe status before being using it for a beneficial purpose. Out of 115 strains, 29 (25%) were negative for blood hemolytic activities. Among these 29 isolates, three isolates did not show a breakdown of gelatin and were recognized as safe. Antibiotic resistance assay showed resistance of two of them against antibiotics discs of Streptomycin (10 µg), Ciprofloxacin (20 µg), Vancomycin (30 µg), Metronidazole (10 µg), Ampicillin (5 µg), Chloramphenicol (30 µg), Kanamycin (30 µg), Erythromycin (15 µg), Penicillin (10 µg) and Tetracycline (30 µg). The bacterial isolate (T-2) was found safe that was identified as Lactobacillus agilis by sequence analysis of 16 s rRNA gene and processed in vitro as an anti-bacterial, anti-oxidant, anti-diabetic, and anti-inflammatory agent. Free radical scavenging activities and inhibition of α-amylase activities for Lactobacillus agilis were found relative to standard drug values as 68% and 73% and 51.3% and 65.3%, respectively. The in-vitro anti-inflammatory assay showed 61.6% (Lactobacillus agilis) while showed 69% (aspirin) activity for denaturation albumin protein. The results suggested that Lactobacillus agilis can be used as a potential probiotic strain as well as can be used to produce nutraceuticals.     

Toxic copper level increases erythrocyte glycolytic rate,glutathione production and alters electrolyte balance in male Wistar rats

BackgroundCopper is a micronutrient vital to several cellular energy metabolic processes and drives erythropoiesis. However, it disrupts cellular biological activities and causes oxidative damage when in excess of cellular needs. This study investigated the effects of copper toxicity on erythrocyte energy metabolism in male Wistar rats.MethodsTen Wistar rats (150–170 g) were randomly divided into 2 groups: control (given 0.1 ml distilled water) and copper toxic (given 100 mg/kg copper sulphate). Rats were orally treated for 30 days. Blood, collected retro-orbitally after sodium thiopentone anaesthesia (50 mg/kg i.p.) into fluoride oxalate and EDTA bottles, was subjected to blood lactate assay and extraction of red blood cell respectively. Red blood cell nitric oxide (RBC NO), glutathione (RBC GSH), adenosine triphosphate (RBC ATP) levels, RBC hexokinase, glucose-6-phosphate (RBC G6P), glucose-6-phosphate dehydrogenase (RBC G6PDH), and lactate dehydrogenase (RBC LDH) activity was estimated spectrophotometrically. Values (Mean±SEM, n = 5) were compared by Student’s unpaired T-test at p < 0.05.Results and conclusionCopper toxicity significantly increased RBC hexokinase (23.41 ± 2.80 µM), G6P (0.48 ± 0.03 µM), G6PDH (71.03 ± 4.76nmol/min/ml) activities, ATP (624.70 ± 57.36 µmol/gHb) and GSH (3.08 ± 0.37 µM) level compared to control (15.28 ± 1.37 µM, 0.35 ± 0.02 µM, 330.30 ± 49.58 µmol/gHb, 54.41 ± 3.01nmol/min/ml and 2.05 ± 0.14 µM respectively, p < 0.05). Also, RBC LDH activity (145.00 ± 19.88mU/ml), NO (3.45 ± 0.25 µM) and blood lactate (31.64 ± 0.91 mg/dl) level were lowered significantly compared to control (467.90 ± 94.23mU/ml, 4.48 ± 0.18 µM and 36.12 ± 1.06 mg/dl respectively). This study shows that copper toxicity increases erythrocyte glycolytic rate and glutathione production. This increase could be connected to a compensatory mechanism for cellular hypoxia and increased free radical generation.     

In vitro antioxidant and cytotoxic activities of polyherbal extracts from Vetiveria zizanioides,Trichosanthes cucumerina,and Mollugo cerviana on HeLa and MCF-7 cell lines

Various metabolites exist in the medicinal plants have lot of potential to cure various diseases and disorders. Plants such as, Vetiveria zizanioides, Trichosanthes cucumerina, and Mollugo cerviana were collected from Western Ghats, Tamilnadu, India. Phytochemicals were extracted from these plants using various organic solvents and tested against Gram-positive and Gram-negative bacteria. The phytochemicals such as, carbohydrate, alkaloids, steroids, saponins, flavonoids and tannin were detected from these medicinal plants. Among the extracts, methanol showed potent activity and this solvent was used to extract polyherbal medicinal plants. Methanol extract of V. zizanioides was found to be highly active against E. coli (27 ± 2 mm), P. mirabilis (19 ± 3 mm) and B. subtilis (18 ± 2 mm). Ethyl acetate extract showed high activity against E. coli (24 ± 2 mm), P. mirabilis (22 ± 3 mm) and B. subtilis (20 ± 1 mm). These three plants were taken at 1:1:1 ratio and extracted with methanol at 1:10 ratio and synergistic activity was tested against bacterial pathogens. Synergistic activity of polyherbal extract was analyzed. The extracted crude herbal medicine was found to be effective against Staphylococcus aureus, E. coli, Enterbacter sp., Pseudomonas aeruginosa, Bacillus subtilis and Proteus mirabilis. The zone of inhibition was 33 ± 3 mm, 17 ± 2 mm, 22 ± 2 mm, 40 ± 2 mm, 33 ± 1 mm and 38 ± 2 mm zone of inhibition against E. coli, S. aureus, P. aeruginosa, P. mirabilis, B. subtilis and Enterobacter sp. Polyherbal extract was found to be highly effective against P. mirabilis and Enterobacter sp. MIC values of polyherbal extract ranged from 29 ± 2.5 µg/ml to 34 ± 2.5 µg/ml. MIC value was found to be less against P. mirabilis and was high against S. aureus. Antioxidant property varied between 49 ± 3% and 95.3 ± 2%. At 20 µg/ml antioxidant activity was reported as 49 ± 3% and it was increased at higher concentrations of polyherbal extract. Two cell lines (HeLa and MCF cell lines) were selected to analyze cytotoxic activity of polyherbal extract. The methanol extract of polyherbal fraction showed cytotoxicity against these two cell lines. The LC50 value was 467 ± 2.9 µg/ml against HeLa cell line and >800 µg/ml against MCF-7 cell lines. The polyherbal extract showed antibacterial, antioxidant and anticancer activities.     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Exposure of preterm neonates to toxic metals during their stay in the Neonatal Intensive Care Unit and its impact on neurodevelopment at 2 months of age

BackgroundPremature neonates might be exposed to toxic metals during their stay in the neonatal intensive care unit (NICU), which could adversely affect neurodevelopment; however, limited evidence is available. The present study was therefore designed to assess the exposure to mercury, lead, cadmium, arsenic, and manganese of preterm neonates who received total parenteral nutrition (TPN) and/or red blood cell (RBC) transfusions during their NICU stay and the risk of neurodevelopment delay at the age of 2 months.MethodsWe recruited 33 preterm neonates who required TPN during their NICU admission. Blood samples were collected for metal analysis at two different time points (admission and before discharge). Metals in the daily TPN received by preterm neonates were analyzed. Neurodevelopment was assessed using the Ages and Stages Questionnaire Edition 3 (ASQ-3).ResultsAll samples of TPN had metal contamination: 96% exceeded the critical arsenic limit (0.3 μg/kg body weight/day); daily manganese intake from TPN for preterm neonates exceeded the recommended dose (1 µg/kg body weight) as it was added intentionally to TPN solutions, raising potential safety concerns. All samples of RBC transfusions exceeded the estimated intravenous reference dose for lead (0.19 µg/kg body weight). Levels of mercury, lead and manganese in preterm neonates at discharge decreased 0.867 µg/L (95% CI, 0.76, 0.988), 0.831 (95%CI, 0.779, 0.886) and 0.847 µg/L (95% CI, 0.775, 0.926), respectively. A decrease in ASQ-3-problem solving scores was associated with higher levels of blood lead in preterm neonates taken at admission (ß = −0.405, 95%CI = −0.655, −0.014), and with plasma manganese (ß = −0.562, 95%CI = −0.995, −0.172). We also observed an association between decreased personal social domain scores with higher blood lead levels of preterm neonates before discharge (ß = −0.537, 95%CI = −0.905, −0.045).ConclusionOur findings provide evidence to suggest negative impacts on the neurodevelopment at 2 months of preterm infants exposed to certain metals, possibly related to TPN intake and/or blood transfusions received during their NICU stay. Preterm neonates may be exposed to levels of metals in utero.     

Cytotoxicity of compounds from Xylopia aethiopica towards multi-factorial drug-resistant cancer cells

IntroductionMultidrug resistance (MDR) in cancer represent a major hurdle in chemotherapy. Previously, the methanol extract of the medicinal spice Xylopia aethiopica displayed considerable cytotoxicity against multidrug resistant (MDR) cancer cell lines.MethodsThe present study was designed to assess the cytotoxicity of compounds, 16α-hydroxy-ent-kauran-19-oic acid (2), 3,4′,5-trihydroxy-6″,6″-dimethylpyrano[2,3-g]flavone (3), isotetrandrine (5) and trans-tiliroside (6) derived from the methanol crude extract of Xylopia aethiopica against 9 drug-sensitive and -resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry.ResultsFlavonoid 3 and alkaloid 5 also displayed IC₅₀ values ranging from 2.61 µM (towards leukemia CCRF-CEM cells) to 18.60 µM (towards gliobastoma multiforme U87MG.ΔEGFR cells) and from 1.45 µM (towards HepG2 cells) to 7.28 µM (towards MDA-MB-231-pcDNA cells), respectively. IC₅₀ values ranged from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Compound 3 induced apoptosis in leukemia CCRF-CEM cells mediated by the disruption of the MMP, whilst 5 induced apoptosis mediated by ROS production.ConclusionsCompounds 2 and 5 represent potential cytotoxic phytochemicals that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.     

Preliminary screening of antioxidant and cytotoxic potential of green seaweed,Halimeda opuntia (Linnaeus) Lamouroux

Marine natural products have displayed numerous advantageous effects on biological activities, including antioxidants and cytotoxicity. The total lipids, carotenoids, chlorophyll a and b content, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of methanolic crude extract of the green seaweed Halimeda opuntia were all measured in this study. The TPC of the extracts was determined according to the Folin-Ciocalteu method, yielding a result of 55.04 ± 0.98 mg GAE/g of extract. As determined by the aluminium chloride colorimetric method, the TFC of the extract was 40.02 ± 0.02 mg QE/g of extract. Antioxidant activity was determined by using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with different concentrations that ranged between 200 and 1000 µg/mL, noted H. opuntia as the highest in DPPH reduction (63.61 %) at 1000 µg/mL concentration. Total antioxidant capacity (TAC) of the extract was 57.36 ± 0.004 mg AAE/g extract at concentration of 1.0 mg/mL. The cytotoxic activity of this seaweed was pre-screened against a panel of cell lines including estrogen receptor-positive human breast adenocarcinoma (MCF-7), estrogen negative human breast adenocarcinoma (MDA-MB-231), human colorectal adenocarcinoma (HT-29), human hepatocellular carcinoma (HepG2), and mouse embryonic fibroblast (3T3) using the MTT assay. The content of total lipids in H. opuntia was 1.60 ± 0.002 %. Total carotenoids were 115.57 ± 0.98 µg/g, while chlorophyll a and b were 148.73 ± 2.60 µg/g and 290.83 ± 9.46 µg/g, respectively. In terms of cytotoxicity activity, methanolic extract of H. opuntia was found to be highly cytotoxic to MCF-7 cells, with an IC₅₀ of 25.14 ± 1.02 g/mL, and slightly less so to 3T3 cells (IC₅₀ 65.23 ± 0.25 µg/mL). This study's findings suggest that natural pigments (carotenoids and chlorophyll), phytochemicals like phenolic and flavonoid compounds found in this species may play an important role and could be used as a natural cancer treatment.     

In vitro assessment of Thimerosal cytotoxicity and antimicrobial activity

BackgroundThimerosal (Merthiolate) is a well-known preservative used in pharmaceutical products, the safety of which was a matter of controversy for decades. Thimerosal is a mercury compound, and there is a debate as to whether Thimerosal exposure from vaccination can contribute to the incidence of mercury-driven disorders. To date, there is no consensus on Thimerosal safety in Vaccines. In 1977, a maximum safe dose of 200 μg/ml (0.5 mM) was recommended for Thimerosal by the WHO experts committee on biological standardization. Up-to-date guidelines, however, urge national control authorities to establish their own standards for the concentration of vaccine preservatives. We believe such safety limits must be studied at the cellular level first. The present study seeks a safe yet efficient dose of Thimerosal exposure for human and animal cells and control microorganism strains.MethodsThe safety of Thimerosal exposure on cells was analyzed through an MTT cell toxicity assay. The viability of four cell types, including HepG2, C2C12, Vero Cells, and Peripheral blood mononuclear cells (PBMCs), was examined in the presence of different Thimerosal concentrations and the maximum tolerable dose (MTD) and the half maximal inhibitory concentration (IC50) values for each cell line were determined. The antimicrobial effectiveness of Thimerosal was evaluated on four control strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis, to obtain the minimum inhibitory concentration (MIC) of Thimerosal. The MIC test was performed in culture media and under optimal growth conditions of microorganisms in the presence of different Thimerosal concentrations.ResultsThe viability of all examined cell lines was suppressed entirely in the presence of 4.6 μg/ml (12.5 μM) of Thimerosal. The MTD for HepG2, C2C12, PBMC, and Vero cells was 2, 1.6, 1, and 0.29 μg/ml (5.5, 4.3, 2.7 and 0.8 μM), respectively. The IC50 of Thimerosal exposure for HepG2, C2C12, PBMC, and Vero cells was 2.62, 3.17, 1.27, and 0.86 μg/ml (7.1, 8.5, 3.5 and 2.4 μM), respectively. As for antimicrobial effectiveness, the growth capability of Candida albicans and Staphylococcus aureus was suppressed entirely in the presence of 6.25 µg/ml (17 μM) Thimerosal. The complete growth inhibition of Pseudomonas aeruginosa in culture media was achieved in 100 µg/ml (250 µM) Thimerosal concentration. This value was 12.5 µg/ml (30 μM) for Aspergillus brasiliensis.ConclusionAccording to our results Thimerosal should be present in culture media at 100 μg/ml (250 µM) concentration to achieve an effective antimicrobial activity. We showed that this amount of Thimerosal is toxic for human and animal cells in vitro since the viability of all examined cell lines was suppressed in the presence of less than 5 μg/ml (12.5 μM) of Thimerosal. Overall, our study revealed Thimerosal was 333-fold more cytotoxic to human and animal cells as compared to bacterial and fungal cells. Our results promote more study on Thimerosal toxicity and its antimicrobial effectiveness to obtain more safe concentrations in biopharmaceuticals.     

Onosma hispidum L. extract reverses hyperlipidemia,hypertension, and associated vascular dysfunction in rats

Onosma hispidum.L (O. hispidum) belongs to the family Boregineacea. A preliminary study and its medicinal use suggested its role in the management of hyperlipidemia. The present study aimed to assess the effect of methanolic root extract of O. hispidum in hyperlipidemia and associated vascular dysfunction. Oral administration of O. hispidum crude extract (Oh. Cr) to tyloxopol and high fat diet-induced hyperlipidemic Sprague-Dawley rats for 10 and 28 days significantly reduced total triglycerides and cholesterol (p < 0.001), compared to hyperlipidemic rats. Oh. Cr 250 mg/kg orally treated rats significantly (p < 0.001) reduced both the total body weight and atherogenic index in tylaxopol and HFD rats. In HMG-CoA assay, the inhibition of the enzyme was significant in Oh.Cr (250 mg/kg) treated group. Histopathological studies indicated that the group treated with Oh.Cr 250 mg/kg/day showed regular morphology of aortic intima, media and adventitia, and improved the endothelial damage. To investigate the vascular dysfunction, isolated rat aorta rings from all groups were pre-contracted with 1 µM phenylephrine (PE), and the effect of acetylcholine (Ach) was monitored. In the aorta isolated from Oh.Cr (50 mg/kg) treated group, Ach completely relaxed the PE-induced contraction with EC₅₀ value of 0.05 µg/mL 0.015 (0.01–0.2) compared to the hyperlipidemic control group (<30% relaxation). In atorvastatin (10 mg/kg) treated rat aorta, Ach showed 50% relaxation. The Oh.Cr extract also reduced (105.92 ± 1.14 to 66.63 ± 0.85 mmHg) mean arterial pressure in hyperlipidemic hypertensive rats. These findings suggest that extract of O. hispidum is an effective remedy for hypercholesterolemia, and hypertriglyceridemia, which acts through inhibition of HMG-CoA and improving vascular dysfunction.     

Cytotoxicity of a naturally occurring furoquinoline alkaloid and four acridone alkaloids towards multi-factorial drug-resistant cancer cells

IntroductionChemotherapy is one of the preferred mode of treatment of malignancies, but is complicated by the expression of diverse resistance mechanisms of cancer cells.MethodsIn the present study, we investigated the cytotoxicity of five alkaloids including a furoquinoline montrofoline (1) and four acridones namely 1-hydroxy-4-methoxy-10-methylacridone (2), norevoxanthine (3), evoxanthine (4), 1,3-dimethoxy-10-methylacridone (5) against 9 drug-sensitive and multidrug-resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry.ResultsFuroquinoline 1 as well as the acridone alkaloids 2–5 displayed cytotoxic effects with IC₅₀ values below 138 µM on all the 9 tested cancer cell lines. The IC₅₀ values ranged from 41.56 µM (towards hepatocarinoma HepG2 cells) to 90.66 µM [towards colon carcinoma HCT116 (p53^−/−) cells] for 1, from 6.78 µM [towards HCT116 (p53^−/−) cells) to 106.47 µM [towards breast adenocarcinoma MDA-MB-231-pcDNA cells] for 2, from 5.72 µM (towards gliobastoma U87MG.ΔEGFR cells) to 137.62 µM (towards leukemia CCRF-CEM cells] for 3, from 6.11 µM [towards HCT116 (p53^+/+) cells] to 80.99 µM (towards HepG2 cells] for 4, from 3.38 µM (towards MDA-MB-231-BCRP cells) to 58.10 µM (towards leukemia CEM/ADR5000 cells] for 5 and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Acridone alkaloid 5 induced apoptosis in CCRF-CEM leukemia cells, mediated by increased ROS production.ConclusionsThe five tested alkaloids and mostly acridone 5 are potential cytotoxic natural products that deserve more investigations to develop novel cytotoxic compounds against multifactorial drug-resistant cancers.     

Synergistic mutual potentiation of antifungal activity of Zuccagnia punctata Cav. and Larrea nitida Cav. extracts in clinical isolates of Candida albicans and Candida glabrata

BackgroundZuccagnia punctata Cav. (Fabaceae) and Larrea nitida Cav. (Zygophyllaceae) are indistinctly or jointly used in traditional medicine for the treatment of fungal-related infections. Although their dichloromethane (DCM) extract have demonstrated moderate antifungal activities when tested on their own, antifungal properties of combinations of both plants have not been assessed previously.PurposeThe aim of this study was to establish with statistical rigor whether Z. punctata (ZpE) and L. nitida DCM extract (LnE) interact synergistically against the clinically important fungi Candida albicans and Candida glabrata and to characterize the most synergistic combinations.Study designFor synergism assessment, the statistical-based Boik's design was applied. Eight ZpE–LnE fixed-ratio mixtures were prepared from four different months of 1 year and tested against Candida strains. L_ϕ (Loewe index) of each mixture at different fractions affected (ϕ) allowed for the finding of the most synergistic combinations, which were characterized by HPLC fingerprint and by the quantitation of the selected marker compounds.MethodsL_ϕ and confidence intervals were determined in vitro with the MixLow method, once the estimated parameters from the dose–response curves of independent extracts and mixtures, were obtained. Markers (four flavonoids for ZpE and three lignans for LnE) were quantified in each extract and their combinations, with a valid HPLC–UV method. The 3D-HPLC profiles of the most synergistic mixtures were obtained by HPLC–DAD.ResultsThree over four IC₅₀ZpE/IC₅₀LnE fixed-ratio mixtures displayed synergistic interactions at effect levels ϕ > 0.5 against C. albicans. The dosis of the most synergistic (L_ϕ = 0.62) mixture was 65.96 µg/ml (ZpE = 28%; LnE = 72%) containing 8 and 36% of flavonoids and lignans respectively. On the other hand, one over four IC₅₀ZpE/IC₅₀LnE mixtures displays synergistic interactions at ϕ > 0.5 against C. glabrata. The dosis of the most synergistic (L_ϕ = 0.67) mixture was 168.23 µg/ml (ZpE = 27%; LnE = 73%) with 9.7 and 31.6% of flavonoids and lignans respectively.Conclusions Studies with the statistical-based MixLow method, allowed for the finding of the most ZpE–LnE synergistic mixtures, giving support to a proper joint use of both antifungal herbs in traditional medicine.     

Cytotoxicity of three naturally occurring flavonoid derived compounds (artocarpesin,cycloartocarpesin and isobavachalcone) towards multi-factorial drug-resistant cancer cells

IntroductionCancer remains an aggressive deadly disease, if drug resistance develops. This problem is aggravated by the fact that multiple rather than single mechanisms are involved in resistance and that multidrug resistance (MDR) phenomena cause inefficacy of many clinical established anticancer drugs. We are seeking for novel cytotoxic phytochemicals to combat drug-resistant tumour cells.MethodsIn the present study, we investigated the cytotoxicity of three naturally occurring flavonoids including two flavones artocarpesin (1) and cycloartocarpesin (2) and one chalcone, isobavachalcone (3) against 9 drug-sensitive and MDR cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analysed via flow cytometry.ResultsFlavones 1 and 2 as well as chalcone 3 displayed cytotoxic effects at various extent on all the 9 tested cancer cell lines with IC₅₀ values respectively below 106 µM, 50 µM and 25 µM. The IC₅₀ values for the three investigational flavonoids ranged from 23.95 µM (towards hepatocarcinoma HepG2 cells) to 105 µM [towards colon carcinoma HCT116 (p53^−/−) cells] for 1, from 15.51 µM (towards leukemia CCRF-CEM cells) to 49.83 µM [towards glioblastoma U87MG.ΔEGFR cells] for 2 and from 2.30 µM (towards CCRF-CEM cells) to 23.80 µM [towards colon carcinoma HCT116 (p53^+/+) cells] for 3 and from 0.20 µM (towards CCRF-CEM cells) to 195.12 µM (towards leukemia CEM/ADR5000 cells) for doxorubicin. Compounds 2 and 3 induced apoptosis in CCRF-CEM leukemia cells, mediated by caspase activation and the disruption of MMP.ConclusionsThe three tested flavonoids and mostly chalcone 3 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.     

Bronchipret® syrup containing thyme and ivy extracts suppresses bronchoalveolar inflammation and goblet cell hyperplasia in experimental bronchoalveolitis

Background/purposeAcute bronchitis (AB) is a common lung condition characterized by inflammation of the large bronchi in response to infection. Bronchipret^® syrup (BRO), a fixed combination of thyme and ivy extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to identify potential mechanisms of action.MethodsBronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o. once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24–72 h post LPS challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO).ResultsBRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular release of LTB₄ (IC₅₀ = 36 µg⋅ml⁻¹) and cysLT (IC₅₀ = 10 µg⋅ml⁻¹) and inhibited the activity of isolated 5-LO (IC₅₀ = 19 µg⋅ml⁻¹).ConclusionBRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may contribute to its observed clinical efficacy in AB.     

Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoidea on human ovarian cancer cell lines

Margaritaria discoidea (Baill.) G. L. Webster (Euphorbiaceae) is a well-known medicinal plant in Africa used for the treatment of various diseases. So far, no cytotoxic effects of plant extracts on cancer cell lines have been reported.Aim of the studyTo evaluate the cytotoxicity against human ovarian cancer cells of extracts of M. discoidea and characterize the major bioactive compounds.MethodsBoth organic and aqueous extracts of this plant were obtained by maceration. The sulforhodamine B cell proliferation assay was used for evaluation of their cytotoxic activities and the potential bioactive compounds were characterized by gas chromatography–mass spectrometry.ResultsThe organic extract of M. discoidea showed stronger cytotoxicity than the aqueous extract with IC₅₀ values of 14.4 ± 3.0, 14.2 ± 1.2 and 34.7 ± 0.5 µg/ml on OVCAR-8, A2780 and cisplatin-resistant A2780cis ovarian cancer cells, respectively. The organic extract was further subjected to bioassay-guided fractionation by partitioning with n-hexane, ethyl acetate, and n-butanol in water. The ethyl acetate fraction was the most potent on the three ovarian cancer cell lines. A GC–MS analysis of trimethylsilyl derivatives of this fraction indicated the presence of phenolic compounds such as gallic acid and the alkaloid securinine. The IC₅₀ values of these two compounds were determined to be in the range of 3–16 µM, which indicated that they could contribute to the cytotoxic activity of the extract of M. discoidea.ConclusionsThis study has evaluated the cytotoxicity of stem bark extracts of M. discoidea against ovarian cancer cells and provided a basis of further development of this plant for the treatment of ovarian cancer.     

Nanoencapsulation of quercetin enhances its dietary efficacy in combating arsenic-induced oxidative damage in liver and brain of rats

AimsThis study was performed to evaluate the therapeutic efficacy of nanocapsulated flavonoidal quercetin (QC) in combating arsenic-induced reactive oxygen species (ROS)-mediated oxidative damage in hepatocytes and brain cells in a rat model.Main methodsHepatic and neuronal cell damage in rats was made by a single injection (sc) of sodium arsenite (NaAsO₂, 13 mg/kg b. wt. in 0.5 ml of physiological saline). A single dose of 500 µl of quercetin suspension (QC) (QC 8.98 µmol/kg) or 500 µl of nanocapsulated QC (NPQC) (QC 8.98 µmol/kg) was given orally to rats at 90 min prior to the arsenite injection.Key findingsInorganic arsenic depositions (182 ± 15.6 and 110 ± 12.8 ng/g protein) were found in hepatic and neuronal mitochondrial membranes. Antioxidant levels in hepatic and neuronal cells were reduced significantly by arsenic. NPQC prevented the arsenite-induced reduction in antioxidant levels in the liver and brain. Arsenic induced a substantial decrease in liver and brain cell membrane microviscosities, and NPQC treatment resulted in a unique protection against the loss. A significant correlation between mitochondrial arsenic and its conjugated diene level was observed both in liver and brain cells for all experimental rats.SignificanceArsenic-specific antidotes are used against arsenic-induced toxicity. However, the target site is poorly recognized and therefore achieving an active concentration of drug molecules can be a challenge. Thus, our objective was to formulate NPQC and to investigate its therapeutic potential in an oral route against arsenite-induced hepatic and neuronal cell damage in a rat model.     

Toxicity of seventeen insecticides to Camponotus sericeus (Hymenoptera: Formicidae)

The success of chemical control depends on toxicity of insecticides against insect pests. Camponotus sericeus is an important urban pest with the ability to cause substantial damage to wooden structures, but there is a lack of information on toxicity of insecticides against C. sericeus. To determine the insecticide toxicity, workers of C. sericeus were exposed to 17 insecticides from different classes: carbamate (methomyl, bendiocarb), organophosphate (chlorpyrifos, profenofos, temephos), pyrethroid (bifenthrin, deltamethrin, permethrin), neonicotinoid (acetamiprid, imidacloprid, thiamethoxam), avermectin (abamectin, emamectin), pyrrole (chlorfenapyr), phenylpyrazole (fipronil), and spinosyn (spinosad and spinetoram), via residual bioassay method. The LC₅₀ ranged from: 0.15 to 0.20 µg/vial for carbamates, 0.09 to 0.27 µg/vial for organophosphates, 0.09 to 0.44 µg/vial for pyrethroids, 0.02 to 0.67 µg/vial for neonicotinoids, 0.54 to 0.82 µg/vial for avermectins, 0.78 µg/vial for pyrrole, 0.62 µg/vial for phenylpyrazole, and 1.96 to 2.05 µg/vial for spinosyns. Overall, acetamiprid was the most toxic one among the tested insecticides followed by permethrin, temephos, profenofos, bendiocarb and methomyl, while spinosad and spinetoram were the least toxic insecticides. Considering the potential toxicity of different insecticides against C. sericeus, future studies could investigate the practical application of these insecticides in order to design an effective management plan.