Characterization, Anti-glycation, Anti-inflammation, and Lipase Inhibitory Properties of Rauvolfia vomitoria Leaf Extract: In Vitro and In Silico Evaluations for Obesity Treatment.

Pancreatic lipase (PLP) is an enzyme responsible for the catalytic hydrolysis of fats and its inhibition is relevant for obesity management. Side effects linked with orthodox inhibitors have, however, paved the way for an increased search for safe natural sources. The present study investigated the anti-glycation, anti-inflammatory, and anti-lipase properties of Rauvolfia vomitoria aqueous (ARV), ethanolic (ERV), and methanolic (MRV) leaf extracts coupled with the molecular interactions of selected bioactive compounds with PLP using in vitro and in silico techniques. Phytochemical constituents were characterized using spectroscopic techniques. Drug-likeness and chemical reactivity profile of selected bioactive compounds were analyzed using SwissADME and quantum chemical calculations. FT-IR and GC-MS affirmed the presence of phenolic compounds including 3-phenyl-2-ethoxypropylphthalimide and 5-methyl-2-phenyl-1H-indole. All extracts showed moderate anti-glycation, anti-inflammatory, and lipase inhibitory capacities relative to standard controls. However, MRV exhibited the highest lipase inhibition (IC50, 0.17 ± 0.01 mg/mL), using a mixed-inhibition pattern. MRV interaction with PLP resulted in decreased secondary structure components of PLP (α-sheet, ß-turn). MRV compounds (MCP20, MCP28, etc.) exhibited low chemical hardness, EHOMO-ELUMO energy gap, and high chemical reactivity. Foremost MRV compounds obeyed Lipinski's rule of five for drug-likeness and interacted with PHE-78 amongst others at PLP catalytic domain with high binding affinity (≥ - 9.3 kcal/mol). Pi-alkyl hydrophobic interaction and hydrogen bonding were predominantly involved. Our findings provide scientific insights into the ethnotherapeutic uses of R. vomitoria extracts for the management of obesity and related complications, plus useful information for optimizable drug-like candidates against obesity.

Solid-state fermentation production of L-lysine by Corynebacterium glutamicum (ATCC 13032) using agricultural by-products as substrate.

To meet the growing demand for L-lysine, an essential amino acid with various applications, it is crucial to produce it on a large scale locally instead of relying solely on imports. This study aimed to evaluate the potential of using Corynebacterium glutamicum ATCC 13032 for L-lysine production from agricultural by-products such as palm kernel cake, soybean cake, groundnut cake, and rice bran. Solid-state fermentation was conducted at room temperature for 72 h, with the addition of elephant grass extract as a supplement. The results revealed that these agricultural by-products contain residual amounts of L-lysine. By employing solid-state fermentation with C. glutamicum (106 CFU/ml) in 100 g of various agricultural by-products, L-lysine production was achieved. Interestingly, the addition of elephant grass extract (1 g of elephant grass: 10 ml of water) further enhanced L-lysine production. Among the tested substrates, 100 g of groundnut cake moistened with 500 ml of elephant grass extract yielded the highest L-lysine concentration of 3.27 ± 0.02 (mg/gds). Furthermore, fermentation led to a substantial rise (p < 0.05) in soluble protein, with solid-state fermented soybean cake moistened with 500 ml of elephant grass extract exhibiting the highest amount of 7.941 ± 0.05 mg/gds. The activities of xylanase, amylase and protease were also significantly enhanced. This study demonstrates a viable biotechnological approach for locally producing L-lysine from agricultural by-products using solid-state fermentation with C. glutamicum. The findings hold potential for both health and industrial applications, providing a sustainable and economically feasible method for L-lysine production.

Solid-state fermentation of cassava (Manihot esculenta Crantz): a review.

Solid-state fermentation (SSF) is a promising technology for producing value-added products from cassava (Manihot esculenta Crantz). In this process, microorganisms are grown on cassava biomass without the presence of free-flowing liquid. Compared to other processing methods, SSF has several advantages, such as lower costs, reduced water usage, and higher product yields. By enhancing the content of bioactive compounds like antioxidants and phenolic compounds, SSF can also improve the nutritional value of cassava-based products. Various products, including enzymes, organic acids, and biofuels, have been produced using SSF of cassava. Additionally, SSF can help minimize waste generated during cassava processing by utilizing cassava waste as a substrate, which can reduce environmental pollution. The process has also been explored for the production of feed and food products such as tempeh and cassava flour. However, optimizing the process conditions, selecting suitable microbial strains, and developing cost-effective production processes are essential for the successful commercialization of SSF of cassava.

Biochemical characterization of solid-state fermented cassava roots (Manihot esculenta Crantz) and its application in broiler feed formulation.

The biochemical parameters of solid-state fermented peeled and unpeeled cassava roots (Manihot esculenta Crantz) and their application in broiler feed formulations were investigated. Fermentation occurred at room temperature for 72 h (pH 3-9). The samples utilized for five (5) broiler starter feeds were labeled: control, unfermented unpeeled cassava (UUC), unfermented peeled cassava (UPC), fermented unpeeled cassava (FUC), and fermented peeled cassava (FPC). Formulations were made by substituting fermented/non-fermented cassava roots at pH 7 for maize (w/w%). Fermentation-induced changes included increased soluble and total protein concentrations (69.3 and 334.5 mg/g) and (9.6 and 10.8%), respectively, in cultures prepared with peeled and unpeeled cassava at pH 7 compared to the control (p < 0.05), and a reduction (p < 0.01) in cyanide concentration from 44.4 to 78.7 mg/kg in the control to 8.5 and 13.7 mg/kg in fermented cassava at pH 7. Birds fed FUC and FPC meal (0.6 and 0.5 kg) gained significantly more weight (p < 0.05) than those fed the control (0.3 kg). The biochemical parameters aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and urea levels in broiler serum did not differ significantly (p > 0.05) for birds fed with fermented peeled and unpeeled cassava. Conversely, serum albumin and calcium levels were significantly lower (p < 0.05) for birds fed with the control feed compared to birds fed with fermented feeds. The results imply that fermented peeled and unpeeled cassava roots could be a safe and nutritionally beneficial replacement for maize in broiler diet.

Biochemical Characterization of Solid-State Fermented Cassava Stem (Manihot esculenta Crantz-MEC) and Its Application in Poultry Feed Formulation.

The utilization of solid-state fermentation (SSF) of cassava stem, "Manihot esculenta Crantz-MEC", is central in this study for its biochemical characterization and formulation of a new poultry feed using a starter culture of Rhizopus oligosporus strain at specified experimental conditions (26 ± 1 °C, 72 h and pH 6). The coupling of R. oligosporus strain to SSF of cassava stem caused significant increase (p < 0.05) in glucose, total reducing sugar (TRS) and total soluble protein (TSP) concentrations at variable but marked effect at 10% inoculum size of the fermented cassava stem, as compared with the unfermented type. Further evaluations of DPPH-radical scavenging activity, total phenolic and flavonoid contents (TPC and TFC), as indices of correlation to antioxidant activity in both fermented and unfermented cassava stems showed marked significant difference with prominence at 10% inoculum size (p < 0.05). Results of high α-amylase activities were observed in fermented cassava stem when compared with the unfermented type (p < 0.05) at increasing inoculum sizes (5-15%) but with marked dominance at 10%. Broiler chicks fed with formulated feed showed marked increase in weight gain at 10% inoculum size of the fermented cassava stem relative to a typical poultry feed. Also, examination of alkaline phosphatase (ALP) and alanine and aspartate aminotransferases (ALT and AST) showed no marked difference in their activities for fermented feed at increasing inoculum sizes when compared with typical poultry feed, respectively (p > 0.05). The study hereby suggests the use of fermented cassava stem as an alternative raw material during formulation of livestock feeds.

Anti-obesity, antioxidant and in silico evaluation of Justicia carnea bioactive compounds as potential inhibitors of an enzyme linked with obesity: Insights from kinetics, semi-empirical quantum mechanics and molecular docking analysis.

Obesity is a global health problem characterized by excessive fat deposition in adipose tissues and can be managed by targeting pancreatic lipase (PL) activity. In the present study, we investigated the in vitro antioxidant and anti-obesity potentials of methanolic leaf extract of Justicia carnea(MEJC) using lipase inhibition kinetics model. In silico evaluations of MEJC bioactive compounds as potential drug-like agents and inhibitors of PL were also investigated using SwissADME prediction tool, semi-empirical quantum mechanics(SQM), molecular electrostatic potential(MEP) and molecular docking analysis. Gas chromatography-mass spectrometry(GC-MS) revealed presence of campesterol, stigmasterol, beta-amyrin etc. MEJC scavenged reactive species and inhibited PL activity via a mixed inhibition pattern (Ki = 107.69 µg/mL; Kii = 398.00 µg/mL) with IC50 > orlistat's IC50. Molecular docking of GC-MS identified compounds with porcine PL showed compounds 8,10,12 and 14 having high PL-binding affinity and similar binding pose with orlistat. Hydrophobic interactions and van der Waals forces were predominantly involved in the ligands' interactions with some key catalytic site amino acid residues (Ser-153,His-264). Compounds 10,12,13 and 14 indicated high drug-likeness, bioavailability, electronegativity, ELUMO-EHOMO energy gaps and MEP. Our findings show that MEJC is a rich natural source of antioxidant and anti-obesity agents which could be optimized for development of new anti-obesity drugs.

Phytochemical profile, antioxidant, α-amylase inhibition, binding interaction and docking studies of Justicia carnea bioactive compounds with α-amylase.

The present study investigated the antioxidant and invitro antidiabetic capacities of Justicia carnea aqueous leaf extract (JCAE) using α-amylase inhibition model. α-Amylase binding-interaction with JCAE was also investigated using fluorescence spectroscopy and molecular docking. Phytochemical screening and Gas Chromatography-Mass Spectrometry (GC-MS) analysis indicated presence of bioactive compounds. Phenolic (132 mg GAE/g) and flavonoid contents (31.08 mg CE/g) were high. JCAE exhibited high antioxidant capacity and effectively inhibited α-amylase activity (IC50, 671.43 ± 1.88 µg/mL), though lesser than acarbose effect (IC50, 108.91 ± 0.61 µg/mL). α-Amylase intrinsic fluorescence was quenched in the presence of JCAE. Ultraviolet-visible and FT-IR spectroscopies affirmed mild changes in α-amylase conformation. Synchronous fluorescence analysis indicated alterations in the microenvironments of tryptophan residues near α-amylase active site. Molecular docking affirmed non-polar interactions of compounds 6 and 7 in JCAE with Asp-197 and Trp-58 residues of α-amylase, respectively. Overall, JCAE indicated potential to prevent postprandial hyperglycemia by slowing down carbohydrate hydrolysis.

Isolation, identification and in silico analysis of alpha-amylase gene of Aspergillus niger strain CSA35 obtained from cassava undergoing spoilage.

In this investigation, a gene (CDF_Amyl) encoding extracellular α-amylase in Aspergillus niger strain CSA35 associated with cassava spoilage was amplified using specific primers and characterized in silico. The gene had a partial nucleotide sequence of 968 bp and encoded a protein of 222 aa residues with a molecular weight and isoelectric point of 25.13 kDa and 4.17, respectively. Its catalytic site was located in the active site domain. BLASTp analysis showed that the protein primary sequence of the α-amylase gene had 98% and 99% homologies with the α-amylase of A. niger and A. oryzae RIB40, respectively. The gene is more closely related to α-amylase genes from fungi than to bacterial, plant, or animal α-amylase genes. Restriction mapping of the gene showed it can be digested with restriction enzymes like NcoI, PstI, SmaI, and BcLI among others but not with EcoRI and EcoRV. Its protein product had a hydrophobicity score of - 0.43 but no transmembrane helix. The CDF_Amyl protein was subcellularly localized in the secretory pathway, an indication of its release into extracellular space after secretion. Also, the 3D structure of the CDF-Amyl protein was barrel-shaped with domains characteristic of α-amylases. The encoded α-amylase Vmax is 6.90 U/mg protein and Km is 6.70 mg/ml. It was concluded that the unique characteristics of the CDF_Amyl gene and its deduced protein could find applications in biotechnological, food and pharmaceutical industries where cloning and further modification of this gene would be required for product development and improvement.

Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes.

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.

Isolation of the carbon catabolite repressor (CREA) gene from the plant-pathogenic fungus Cochliobolus carbonum.

The CREA gene has been implicated in glucose repression in several fungi. The product of this gene, CreA, binds to the promoter region of several enzymes and down-regulates gene expression. An ortholog of CREA was isolated and characterized from the maize pathogenic fungus, Cochliobolus carbonum. The deduced amino acid sequence of the C. carbonum CREA gene is very similar to the CreA proteins of Aspergillus niger, Gibberella fujikuroi, Sclerotinia sclerotiorum and Trichoderma reesei, as well as the Mig1 protein of Saccharomyces cerevisiae. And like the other fungal proteins, C. carbonum CreA has two zinc finger regions and a nuclear localization signal. Putative CreA binding sequences were also identified in the 5' region of three C. carbonum cell wall degrading enzyme genes suggesting that the protein may play a role in the regulatory process that controls these enzymes expression.