A novel temperature stable alkaline protease yielding bacteria was isolated from the soils of Dachigam National Park, which is known to be inhabited by a wide variety of endemic plant and animal species of Western Himalaya. This high-potential protease producing isolate was characterized and identified as Bacillus amyloliquefaciens strain HM48 by morphological, Gram’s staining and biochemical techniques followed by molecular characterization using 16S rRNA approach. The extracellular protease of B. amyloliquefaciens HM48 was purified by precipitating with ammonium sulfate (80%), followed by dialysis and Gel filtration chromatography increasing its purity by 5.8-fold. The SDS–PAGE analysis of the purified enzyme confirmed a molecular weight of about ≈25 kDa. The enzyme displayed exceptional activity in a broad temperature range (10–90 °C) at pH 8.0, retaining its maximum at 70 °C, being the highest reported for this proteolytic Bacillus sp., with KM and Vmax of 11.71 mg/mL and 357.14 µmol/mL/min, respectively. The enzyme exhibited remarkable activity and stability against various metal ions, surfactants, oxidizing agent (H2O2), organic solvents and displayed outstanding compatibility with widely used detergents. This protease showed effective wash performance by exemplifying complete blood and egg-yolk stains removal at 70 °C and efficiently disintegrated chicken feathers making it of vital importance for laundry purpose and waste management. For functional analysis, protease gene amplification of strain HM48 yielded a nucleotide sequence of about 700 bp, which, when checked against the available sequences in NCBI, displayed similarity with subtilisin-like serine protease of B. amyloliquefaciens. The structure of this protease and its highest-priority substrate β-casein was generated through protein modeling. These protein models were validated through futuristic algorithms following which protein–protein (protease from HM48 and β-casein) docking was performed. The interaction profile of these proteins in the docked state with each other was also generated, shedding light on their finer details. Such attributes make this thermally stable protease novel and suitable for high-temperature industrial and environmental applications.
Withania coagulans (W. coagulans) is well-known in herbal medicinal systems for its high biological potential. Different parts of the plant are used against insomnia, liver complications, asthma, and biliousness, as well as it is reported to be sedative, emetic, diuretic, antidiabetic antimicrobial, anti-inflammatory, antitumor, hepatoprotective, antihyperglycemic, cardiovascular, immuno-suppressive and central nervous system depressant. Withanolides present in W. coagulans have attracted an immense interest in the scientific field due to their diverse therapeutic applications. The current study deals with chemical and biological evaluation of chloroform, and n-butanol fractions of W. coagulans. The activity-guided fractionation of both extracts via multiple chromatographic steps and structure elucidation of pure isolates using spectroscopies (NMR, mass spectrometry, FTIR and UV-Vis) led to the identification of a new withanolide glycoside, withacogulanoside-B (1) from n-butanol extract and five known withanolides from chloroform extract [withanolid J (2), coagulin E (3), withaperuvin C (4), 27-hydroxywithanolide I (5), and ajugin E (6)]. Among the tested compounds, compound 5 was the most potent α-glucosidase inhibitor with IC50 = 66.7 ± 3.6 µM, followed by compound 4 (IC50: 407 ± 4.5 µM) and compound 2 (IC50: 683 ± 0.94 µM), while no antiglycation activity was observed with the six isolated compounds. Molecular docking was used to predict the binding potential and binding site interactions of these compounds as α-glucosidase inhibitors. Consequently, this study provides basis to discover specific antidiabetic compounds from W. coagulans.
Ifosfamide is a widely used chemotherapeutic agent having broad-spectrum efficacy against several tumors. However, nephro, hepato, neuro cardio, and hematological toxicities associated with ifosfamide render its use limited. These side effects could range from organ failure to life-threatening situations. The present study aimed to evaluate the attenuating efficiency of Berberis vulgaris root extract (BvRE), a potent nephroprotective, hepatoprotective, and lipid-lowering agent, against ifosfamide-induced toxicities. The study design comprised eight groups of Swiss albino rats to assess different dose regimes of BvRE and ifosfamide. Biochemical analysis of serum (serum albumin, blood urea nitrogen, creatinine, alanine transaminase, aspartate transaminase, alkaline phosphatase, lactate dehydrogenase, total cholesterol, and triglycerides) along with complete blood count was performed. Kidney, liver, brain, and heart tissue homogenates were used to find malondialdehyde, catalase, and glutathione S-transferase levels in addition to the acetylcholinesterase of brain tissue. The results were further validated with the help of the histopathology of the selected organs. HeLa cells were used to assess the effect of BvRE on ifosfamide cytotoxicity in MTT assay. The results revealed that pre- and post-treatment regimens of BvRE, as well as the combination therapy exhibited marked protective effects against ifosfamide-induced nephro, hepato, neuro, and cardiotoxicity. Moreover, ifosfamide depicted a synergistic in vitro cytotoxic effect on HeLa cells in the presence of BvRE. These results corroborate that the combination therapy of ifosfamide with BvRE in cancer treatment can potentiate the anticancer effects of ifosfamide along with the amelioration of its conspicuous side effects.
As an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4–30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5–35 °C and pH 6.0–12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low Km value of 1.0 mg/ml and Vmax 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.
The present study focuses on the use of n-hexane, chloroform, ethyl acetate and aqueous extracts of Pteris cretica to confirm its in-vivo anti-inflammatory properties and in-vitro cytotoxic profile. The anti-inflammatory study was conducted by carrageenan-induced edema test at different doses of Pteris cretica extracts (250 and 500mg/kg body weight). At 250 mg/kg dose of chloroform, n-hexane, aqueous and ethyl acetate extracts expressed maximum anti-inflammatory activity at the 2 nd hour with 29.65, 29.06, 27.55 and 26.04% respectively by inhibiting carrageenaninduced edema. Moreover, 500 mg/kg concentration of chloroform extract of Pteris cretica expressed efficient anti-inflammatory activity with 45.3% of inhibition in edema; whereas n-hexane, aqueous and ethyl acetate extracts inhibit the edema at 35.34, 29.06 and 27.55% respectively. MTT assay was used to determine the cytotoxic potential of Pteris cretica extracts on HeLa, and BHK-21 cell lines. Chloroform and aqueous extracts expressed the cytotoxic activity with IC50 of 31.48 and 34.26 µg/mL respectively against HeLa cell lines. However, both chloroform and aqueous extracts exhibit IC50 108.50 and 55.76 µg/mL against BHK-21 cell line respectively. Chloroform extract is less cytotoxic against BHK-21 cell line as compared to aqueous extract. Ethyl acetate extract was found to be toxic for both cell lines. Based upon the above results, isolation, identification and purification of phytochemicals from Pteris cretica extracts is highly recommended as a possible extension of the current work.
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