Parasitic diseases like leishmaniasis are major worldwide health problem. Suboptimal therapies and emergence of resistance demands the exploration of all possible sources to find a new drug against leishmaniasis. In this search, endophytes have emerged as an outstanding source of high metabolic diversity. These microorganisms have recently attracted increased attention in the quest of pharmaceutically important compounds. Present study describes the biological evaluation of five endophytic fungi Plectania milleri NFL1, Trichoderma asperellum NFL2, Paraconiothyrium sp. NFL6, Mucor hiemalis NFW6 and Epicoccum nigrum NFW7 isolated from Taxus fuana of West Himalayan region of Pakistan followed by isolation and identification of compounds from selected endophytic fungi with prime focus on their antileishmanial activity. Endophytic fungal strains were initially cultivated on four solid state media (PDA, SDA, modified taxol medium and rice) and extracted with organic solvent ethyl acetate. The crude extracts obtained after extraction, were evaluated in phytochemical assays to determine the total phenolic and total flavonoid content. Biological activities of the extracts were determined by three antioxidant assays (total antioxidant capacity, reducing power and DPPH free radical scavenging assay), antibacterial assay against 8 bacterial strains, antileishmanial assay against two Leishmania sp. i.e. L. tropica and L. amazonensis and anticancer assays by evaluating the inhibition of NFƙB and K-Ras. Cytotoxic potential was evaluated against four human cell lines i.e. prostate cancer cell line PC-3, human colon adenocarcinoma cell line HT-29, estrogen receptor negative human breast cancer cell line MDA-MB-231 and breast cancer cell line MCF-7. Endophytic fungi showed variable biological activities with apparent effect of media used for fermentation. The highest amount of gallic acid equivalent phenolic and quercetin equivalent flavonoid content was found in M. hiemalis NFW6 and E. nigrum NFW7. While significant total antioxidant activity, total reducing power and DPPH scavenging activities were also exhibited by these two strains. Noteworthy antimicrobial activities were exhibited by P. milleri NFL1 and Paraconiothyrium sp. NFL6 particularly against S. epidermidis with zone of inhibition of 20 ± 0.87 and 20.7 ± 1.26 mm, respectively. Results for antileishmanial activities were pronounced xiiifor P. milleri NFL1 and M. hiemalis NFW6 against Leishmania sp. with IC 50 values of 1.5 ± 1.1 and 3.72 ± 1.7 μg/ml, respectively. Significant inhibition of NF-κB signaling pathway (>70 %) was exhibited by P. milleri NFL1 and E. nigrum NFW7. Similarly, P. milleri NFL1 and T. asperellum NFL2 showed pronounced K-Ras inhibition (>60 %). Endophytic fungi expressed significant cytotoxic activities against all the cancerous cell lines except human colon adenocarcinoma cell line HT-29. Most promising results were observed against breast cancer cell line MCF-7 where P. milleri NFL1, T. asperellum NFL2 and Paraconiothyrium sp. NFL6 expressed greater than 75 % inhibition. P. milleri NFL1 was the only strain which showed cytotoxic activity against three tested cell lines i.e. PC-3, MCF-7 and MDA-MB-231. Biological screening was followed by selection of P. milleri NFL1, M. hiemalis NFW6 and Paraconiothyrium sp. NFL6 for scale up fermentation and compound isolation. Crude extracts, obtained after the large scale fermentation of endophytic fungi, were fractionated by normal phase chromatography and subjected to antileishmanial assay for prioritizing fractions for compound isolation. Structure elucidation was done by 1D, 2D NMR along with mass spectrometry. As a result, three known compounds were isolated from P. milleri NFL1; pestalotin (L1F5F7F4), its analogue (L1F3F7) and galiellalactone (L1F4F4F4). M. hiemalis NFW6 resulted in the isolation of a single compound i.e. triolein (W6F4F4) while Paraconiothyrium sp. NFL6 also afforded one compound pachybasin (L6F8F14). All the compounds were evaluated for their antileishmanial and anticancer potential. Galiellalactone (L1F4F4F4) was the only compound with antileishmanial and anticancer activity among all the isolated compounds. Antileishmanial potential of galiellalactone has been reported in this study for the first time. In silico tools were employed in this study to understand the behavior of endophytic fungi produced compound galiellalactone (L1F4F4F4) with Leishmania proteins. Thorough study of the literature resulted in selection of three target proteins i.e. leishmanolysin, trypanothione reductase and cysteine protease in Leishmania. These proteins were selected on the basis of their essentiality for Leishmania and absence from human host. Unavailability of experimentally determined structure led to homology modelling of the selected proteins by using eight web-based servers. Comparative analysis was performed to select the most reliable protein model. xivIn silico evaluation of galiellalactone (L1F4F4F4) against leishmanial targets was completed by docking studies. The docking simulation between the ligand (L1F4F4F4) and target proteins was performed using AutoDock. Ligand-protein complex among all the three target protein was most stable in case of cysteine protease with hydrogen bond and electrostatic interactions and highest binding affinity of −6.5 kcal/mol. These findings give insights into possible action of galiellalactone against Leishmania. The Himalayan region of Pakistan has a huge biodiversity of medicinal plants including Taxus species. Antileishmanial potential of endophytes associated with Taxus fuana from this region has not been previously reported. These findings will highlight the bioactive potential of endophytic fungi and also act as a cornerstone for lead compounds for drug discovery and development.
Linguists and Muslim jurists have depended heavily on the Arabic language in their researches about Islamic legislation. Their writings show a deep relationship between Islamic commandments and the rules of Arabic grammar. The three chapters of this research present a study of their work syntactically, and analytically to explore the earliest approaches and their various dimensions, and to detect the relationship with the Islamic commandments in the current time. The first chapter is about the role of nouns in deriving the Islamic commandments from the Quranic ayaat. The second chapter discusses the role of various verbs in deriving the Islamic commandments from the Quranic Ayaat. The third chapter is about the role of Arabic letters in deriving Islamic commandments from the Quranic Ayat. This study hopes to help not only the linguists of Arabic language but also the scholars of other related fields.
Economically, Pakistan is an agricultural country as the agriculture sector offers employment to 42.3% of the country?s citizens involved with any kind of labor for their livelihood, 19.5% of its gross domestic product (GDP) comes from the agriculture sector apart from the raw materials for many other value-added sectors contributing to huge quantities of added indirect income; livestock sector as for instance, adding 58.33% to the worth of agriculture besides contributing 11.39% directly to GDP. Population of livestock are cattle 44.4, Buffalo 37.7, Sheep 30.1, goat 72.2, Camel 1.1, Horses 0.4, Asses 5.2, Mules 0.2 millions. Asses population increased from 4.9 to 5.2 million while population of Camel, Horses and mules remained unchanged since last year. Horses along with mules and donkeys facing many managerial, nutritional, noninfectious and infectious diseases which decreases production and performance, out of which infectious diseases such as Glanders is life threatening and pose zoonotic potential to human beings specially care takers, veterinarian and public health. To monitor threat due to these asymptomatic carriers to animals and human beings and from places having decreases frequency of disease Glanders in equines, it is unavoidable to develop and optimize a highly specific and sensitive molecular test such as PCR, required to decrease the number of results which are false negative and false positive findings besides being fast and accurate. 200 nasal and blood samples of working equines (horses, mules and donkeys) were collected from the Bahawalpur, Faisalabad, Gujranwala, Lahore and Multan at the sites of Brook hospital. All the samples were collected and transported to the WTO Quality Control Laboratory of UVAS, Lahore keeping in account the all microbiological hygienic and hazardous measures according to OIE protocols. All the animals were injected mallein PPD intra-dermally into the lower eyelid with the result noted after first and second day. Blood of animals were used for DNA extraction by commercially available DNA extraction kit. The extracted DNA were subjected to Gel Electrophoresis for integrity of the extracted DNA and then used further for amplification of target region of the DNA. The extracted DNA was used for the amplification of Flip IS407A gene region using the specific primers. Products of the PCR were visualized with UV light after electrophoresis of the gel and B. mallei was detected in only positive control. The study found high prevalence of the disease and existence of active nasal shedders in equines. The findings implicate importance of targeted surveillance in glanders suspected equines.