Myxobolus spp. (Cnidaria: Myxobolidae) in the circulating blood of fishes from Goiás and Mato Grosso States, Brazil: case report

Myxosporidiosis is an infectious disease caused by myxozoans of the Phylum Cnidaria, Class Myxosporea, and Order Bivalvulida, considered a common parasite in fresh and saltwater fishes that parasitize many organs, especially gills. In the present study, 49 specimens of fishes belonging to eight genera: Tetragonopterus, Leporinus, Myleus, Pirinampus, Rhapiodon, Pygocentrus, Ageneiosus, and Serrasalmus were collected and blood smears were made, fixed with absolute methanol, and stained with Giemsa 10% to survey hemoparasites. However, myxospores were found in the circulating blood of five (10.20%) fishes belonging to genus Tetragonopterus, Myleus, and Pygocentrus. Two morphological types of Myxobolus spp. were identified in all the five fish specimens analyzed. Usually, investigations on myxozoans in fish are carried out with the search for plasmodia or cysts in the fish organs and observation of the cavity of organs. Nevertheless, this study highlights the importance of also examining the blood of these animals, since these parasites can cause severe pathogenic diseases in fish. Thus, the blood analyses can proportionate preventive sanitary control for commercial fish avoiding economic loss.

Extraction and Identification of Phenolic Compounds from the Iraqi Heliotropium europaeum L. plant

The plants of genus Heliotropium L. (Boraginaceae) are well-known for containing the toxic metabolites called pyrrolizidine alkaloids (PAs) in addition to the other secondary metabolites. Its spread in the Mediterranean area northwards to central and southern Europe, Asia, South Russia, Caucasia, Afghanistan, Iran, Pakistan, and India, Saudi Arabia, Turkey, and over lower Iraq, Western desert. The present study includes the preparation of various extracts from aerial parts of the Iraqi plant. Fractionation, screening the active constituent, and identification by chromatographic techniques were carried out.Heliotropium europaeum herbs were first defatted with n-hexane then extracted exhaustively by soxhlet apparatus using absolute methanol. The extract was filtered and the solvent was evaporated by applying a reduced pressure by a rotary evaporator. The residue suspended in distilled water and partitioned with chloroform, ethyl acetate, n-butanol. The hydrolysis step was done for the two fractions (n-butanol and ethyl acetate). Phytochemical analysis for the screening and identification of bioactive substances of the Heliotropium europaeum plant was done for each fraction. The identification of n-butanol and ethyl acetate fractions was carried out by thin-layer chromatography (TLC) and HPLC technique. For quantitive analysis, the concentration was calculated by serial concentrations of external standard materials to build a calibration curve between concentration and its equivalent peak area. The outcomes of this study were the identifications of new six phenolic compounds from H. europaeum ethyl acetate fraction, which exhibited wide biological activity. The identified compounds were kaempferol (1), Silybin (2), caffeic acid (3), Genistein (4), Apigenin (5), in addition to syringic acid (6). In the present study, we regard the first to report such results about the phenolic compounds in H. europaeum extract. A total of six discovered phenolics were identified in this extract for the first time. Our results on H. europaeum constituents provide a scientific base to examine the pharmacological effects of this plant in the future.

Hemoglobin Precipitation: An Index of In Vitro Vasoconstrictive Activities of Methanol Leaf Extracts of Croton megalocarpus Hutch and Lantana camara Linn

The function of innate hemostasis aids the body in bleeding control, preventing the loss of excessive amounts of blood following low-degree injuries. However, injuries of a higher degree may require extrinsic intervention to stop life-threatening blood loss. Astringent agents’ actions result in mechanical constriction of small blood vessels and shrinkage of body tissues, thereby stopping blood loss. This enhances the primary phase of hemostasis, where vasoconstriction is the main mechanism at play during the initial response to injury. The effects of plant extracts on protein precipitation have been linked to blood vessel vasoconstriction. Traditionally, the leaves of Croton megalocarpus Hutch and Lantana camara Linn plants are used by communities living in Makueni County, Kenya, for peripheral bleeding control. However, the effects of extracts of both plants on hemoglobin precipitation have not been evaluated scientifically. In the current study, the activities of methanol extracts of C. megalocarpus (H.) and L. camara (L.) on blood protein precipitation were investigated. The leaves were harvested, cleaned, air-dried, milled, and extracted in absolute methanol before being concentrated into dry powders. A qualitative phytochemical screen revealed the presence of terpenoids, steroids, tannins, phenols, flavonoids, reducing sugars, cardiac glycosides, and carbohydrates in the methanol extract of C. megalocarpus (H.). The methanol extracts of L. camara (L.) contained cardiac glycosides, saponins, tannins, phenols, terpenoids, reducing sugars, and carbohydrates. The hemoglobin precipitation ability of various concentrations of extracts using mice samples was presented as relative astringency following the tannic acid external standard method. Methanol extracts C. megalocarpus (H.) and L. camara (L.) had significantly higher relative astringency compared with the normal control, indicating a protein precipitating activity. The relative astringency observed in both plant extracts is linked to the activity of tannins, phenols, flavonoids, and saponins detected during preliminary phytochemical screening.

Comparative In vitro Antibacterial Properties of Methanol Extracts and Fractions of Myristica fragrans Seed and Thymus vulgaris Leaf

The aim of this work was to compare the antibacterial properties of methanol extracts and fractions of Myristica fragrans seed and Thymus vulgaris leaf on the gram positive and negative bacteria. The Myristica fragrans seeds were crushed, defatted and air-dried. The defatted seed and leaf powders were separately macerated in absolute methanol for 72 hours. The methanol extracts and fractions were reconstituted at different concentrations of 100mg/mL, 80mg/mL, 60mg/mL, 40mg/mL and 20mg/mL for the antibacterial assay by agar diffusion method with activated cultured Staphylococcus aureus and Escherichia coli , incubated at 37oC for 24 hours . The results showed that these plants possess antibacterial activity on the basis of their zones of inhibition. Methanol extract of M. fragrans had a higher activity of 8-19mm on S. aureus than E. coli with 5-14mm range respectively. Ethylacetate fraction had the highest activity with 9-25mm on S. aureus, while chloroform fraction had the highest activity on E. coli with 8-18mm. For T. vulgaris, the methanol extract had a higher activity of 6-18mm on E. coli than S. aureus of 4-17mm and for the fractions, n-hexane fraction had the highest activity of 7-20mm on S. aureus , while aqueous fraction had the highest activity of 5-18mm on E. coli, compared with zones of inhibition of 18mm against S. aureus and 28mm against E. coli for gentamycin of 2mg/mL which was the reference drug. Methanol extracts and fractions of M. fragrans seed and T. vulgaris leaf showed excellent activities on the gram positive and gram negative bacteria but the M. fragrans had a better activity than T. vulgaris.

Evaluation of Immunomodulatory Activity of Extract and Fractions of Sida acuta Burm. f. Leaves in Mice and GC-MS Analysis of n- Hexane Fraction

The leaves of Sida acuta Burm. f. (Malvaceae) has been reported to possess potent anti- inflammatory, anti- plasmodial and anti-microbial activities. The relationship of these bioactivities and immune responses lead to the evaluation of the immunomodulatory activity of Sida acuta Burm. f. leave extract and fractions. This our study was done to determine the immunomodulatory activity and chemical study of methanol leave extract and fractions of Sida acuta Burm. f. The immunomodulatory evaluation was done by invivo Delay Type Hypersensitivity reaction (DTHR) in the body and in vitro measurement of phagocytosis of killed Candida albicans by the phagocyte polymorphonuclear leucocytes using slide method. Acute toxicity, phytochemical and GC-MS analysis were also performed. The DTHR tested in the blood with T-cells in mice showed that the extract and its fractions caused a delayed hypersensitivity response in 24hrs which was very significant (P ? 0.05) in the n- hexane fraction of the extract when compared to the control group at the dose of 100mg/kg. The in vitro studies showed a very significant difference (P ? 0.05) in the positive control group (LEVA) at concentration of 50, 100 and 200µg/ml, in crude extract (SrE) at concentrations of 50, 100 and 200µg/ml, n- hexane fraction 50, 100 and 200µg/ml, Ethyl acetate fraction at 200µg/ml and Absolute methanol fraction at 100µg/ml and also have high percentage phagocytic stimulation (PPS). The acute toxicity test did not cause clinical signs or death within 24hours post treatment in all doses tested and highest dose of 5000mg/kg. Phytochemical analysis revealed the presence of flavonoids, saponins, alkaloids, triterpenoids, tannins, steroids and cardiac glycosides. GC-MS analysis of fraction with highest activity was carried out on n-hexane fraction which showed the presence of some compounds like hexadecanoic acid, 2-hydroxy-1 (hydroxymethyl) ethyl ester, 3,4-seco-5alpha-cholestan-3-oic acid,4-hydroxy-4-methyl epsilon-lacto

Effects of Phytolacca decandra on the viability of murine breast adenocarcinoma (4T1 cells) in vitro

Background: Comparative studies in cancer patients using conventional and alternative therapy have demonstrated that Phytolacca decandra in homeopathic potencies increases survival and improves quality of life of patients bearing breast cancer. In vitro studies show the induction of apoptosis pathways in MCF-7, a human breast cancer cells lineage, after treatment with Phytolacca decandra in different homeopathic dilutions (from 30C to 10M). Recently, we observed significant growth reduction of Ehrlich carcinoma in mice treated with Phytolacca decandra 30cH. Aims: To evaluate Phytolacca decandra effect in different homeopathic dilutions on the phenotypic features, apoptosis index, and cell morphology of 4T1 cells (murine carcinoma cell lineage) in vitro. Method: The potencies 6, 12, 30 and 200 CH prepared in sterile pure water were studied. Dynamized sterile pure water was used as control. The cytotoxicity was evaluated after different cell treatments in culture bottles (25ml) with the homeopathic medicines (equal to 10% of total medium volume). Cells were cultured in a cell density of 5 x 105 cells / ml, treated with the respective potency and, after 24 hours, analyzed for the apoptosis index using Annexin V kit and measured using the Countess® System. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. Cells were previously adhered to a glass cover slip and fixed with absolute methanol. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. Results and discussion: The results obtained up to now show that the treatment with Phytolacca decandra 200cH induced increase of apoptosis index in relation to the control. Moreover, morphological changes were observed in the respective cell smears: the presence of multinucleated cells, some of them presenting up to 8 nuclei and the increase of eosinophilic staining pattern of cytoplasm, even in mononucleated cells. Conclusion: The increase in apoptosis index reproduced the results described in the literature with other cell lineages, but the changes in morphology still deserve further evaluation.

Development and Optimisation of Solid-Phase Extraction of Extractable and Bound Phenolic Acids in Spelt (Triticum spelta L.) Seeds

A solid-phase extraction (SPE) technique was developed and optimised for isolation and concentration of extractable and bound phenolic acids from germinated spelt seeds, for analysis by liquid chromatography–mass spectrometry. Samples initially underwent solvent extraction under different conditions to maximise the yield of phenolic antioxidants. Optimal extraction conditions for extractable phenolics were absolute methanol as solvent, sample-to-methanol ratio 1:9, and reconstitution in non-acidified water. The bound phenolics were extracted from sample pellets using hydrolysis with 2 M NaOH, acidification of the hydrolysate with formic acid, and simultaneous isolation and purification using Strata X polymeric RP tubes. Compared to liquid–liquid extraction, this direct SPE protocol has significant advantages in terms of higher extraction efficiencies of total and individual phenolics and their antioxidant activities. These data suggest that direct SPE represents a rapid and reliable method for quantitative analysis of both the extractable and the commonly overlooked bound phenolics in Triticum spelta seeds.

Condensation Reactions of Methyl Derivatives of Quinoxaline-1,4-Dioxide with 4,4'-Biphenyl Carboxaldehyde

Aims: To synthesis new compounds via condensation reactions between 2-methyl quinoxaline-1,4-dioxide derivatives 4,4'-biphenyl carboxaldehyde. Methodology: The Quinoxalines derivatives were prepared from 2-nitroaniline derivatives using the Beirut reaction, and the condensation reaction was carried out at room temperature in absolute methanol. Based on IR and NMR spectroscopic techniques, the structures of all products have been suggested. For their synthesis, suitable mechanisms have been suggested. Results: In this work, condensation reactions involving 2-methyl quinoxaline-1,4-dioxide derivatives and 4,4'-biphenyl carboxaldehyde were performed. Conclusion: The final compounds, we suppose, have considerable applications in fluorescent and chromophoric activities. In all known solvents, the products were just slightly soluble. Products have been subjected to sulfonation reactions, although with limited success.

The Investigation of Some Phytochemical Compounds Found in Anchusa strigosa L. Grown Naturally in Iraq

Anchusa strigosa L.: Hardy annual biennial or perennial herb, with hairs especially on the leaves., flowers generally regular. Commonly named (Lisan Althour) in Iraq, from Boraginaceae family. The plant contains phenolic acids, flavonoids, alkaloids, sterols, and terpenoids. The Whole plant part defatted with n-hexane for 24 hours. The defatted plant material extracted using absolute methanol by Soxhlet apparatus for 24 hours, the extract fractionated by solvents of different polarity: petroleum ether- chloroform - ethylacetate- and n-butanol respectively. The n-butanol fraction hydrolyzed with 10% HCl for 5 hours by reflex to break down the glycosidic linkage. Rosmarinic acid, caffeic acid, genistein, and silybin were isolated from ethyl acetate fraction by preparative layer chromatography which identified by high performance liquid chromatography HPLC, Fourier transforms infrared (FTIR) spectra, thin- layer chromatography TLC and melting point. Since the plant contain alkaloids so acid- base extraction performed for crude extract resulting from the maceration of the plant parts in methanol (cold method) to obtain the alkaloid that isolated by preparative layer chromatography and then identified by Fourier transforms infrared (FTIR) spectra and thin-layer chromatography (TLC). The aim of this research was to carry out a phytochemical study of this plant since no previous phytochemical investigation work had been done on this species in Iraq.

In-vitro evaluation of antioxidant and antiradical potential of successive extracts, semi-purified fractions and biosynthesized silver nanoparticles of Rumex vesicarius

The aim of the present study was to assess in vitro the antiradical and antioxidant activities of successive extracts and semi-purified fractions from Rumex vesicarius L. In the present work, three extracts (n-Hexane, ethyl acetate and methanol) and 22 column fractions of methanolic extract (as promising extract) were evaluated against 2,2-diphenyl- 1-picrylhydrazyl (DPPH•) and 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) radical scavenging methods as antiradical and antioxidant activities compared with Butylated hydroxytoluene (BHT) as synthetic standard and silver nanoparticles of methanolic extract (Ag-NPs-Me), in addition to analysis of chemical constituents of extract and fraction using Gas chromatography–mass spectrometry (GC-MS). The obtained results revealed that, both methods go parallel showing that the concentration of extract and incubation time are dependent and proportional with phenolic compounds concentration. Absolute methanol extract recorded the highest antioxidant activity when compared with the other crude extracts with 79.3 and 78.8% against DPPH and ABTS respectively when compared with BHT as synthetic standard (89.4 and 89.9%) against DPPH and ABTS respectively. Calculation of the antiradical activity units showed the highest values of methanolic extract and its promising fraction (No. 12) after 300 seconds (5 minutes) comparing with antioxidant activity (30 min). Also, the antioxidant activity increased with synthetic Ag-NPs-Me when compared with methanolic extract by (IC50= 53.9 and 74.6 µg/ml respectively). Thus, the GC-MS analysis of successive extracts of R. vesicarius L showed a highly complex profile, containing approximately 24 different components. One pure compound was identified from fraction No. 12. The identified compound was l-(+)- ascorbic acid 2, 6-dihexadecanoate. The data also revealed presence of closely similar antioxidant activities in methanolic extract or its pure compounds with BHT when mixed at different proportions. From the obtained results it could be concluded that R. vesicarius methanolic extracts and fractions can be extensively used in the production of potential antioxidant, antiradical and AgNPs-Me for biomedical application on the consumer’s health.