Iso-mukaadial acetate and ursolic acid acetate inhibit the chaperone activity of Plasmodium falciparum heat shock protein 70-1.

Plasmodium falciparum is the most lethal malaria parasite. The present study investigates the interaction capabilities of select plant derivatives, iso-mukaadial acetate (IMA) and ursolic acid acetate (UAA), against P. falciparum Hsp70-1 (PfHsp70-1) using in vitro approaches. PfHsp70-1 facilitates protein folding in the parasite and is deemed a prospective antimalarial drug target. Recombinant PfHsp70-1 protein was expressed in E. coli BL21 cells and homogeneously purified by affinity chromatography. The interaction between the compounds and PfHsp70-1 was evaluated using malate dehydrogenase (MDH), and luciferase aggregation assay, ATPase activity assay, and Fourier transform infrared (FTIR). PfHsp70-1 prevented the heat-induced aggregation of MDH and luciferase. However, the PfHsp70-1 chaperone role was inhibited by IMA or UAA, leading to both MDH and luciferase's thermal aggregation. The basal ATPase activity of PfHsp70-1 (0.121 nmol/min/mg) was closer to UAA (0.131 nmol/min/mg) (p = 0.0675) at 5 mM compound concentration, suggesting that UAA has no effect on PfHsp70-1 ATPase activity. However, ATPase activity inhibition was similar between IMA (0.068 nmol/min/mg) (p < 0.0001) and polymyxin B (0.083 nmol/min/mg) (p < 0.0001). The lesser the Pi values, the lesser ATP hydrolysis observed due to compound binding to the ATPase domain. FTIR spectra analysis of IMA and UAA resulted in PfHsp70-1 structural alteration for ß-sheets shifting the amide I band from 1637 cm-1 to 1639 cm-1, and for α-helix from 1650 cm-1 to 1652 cm-1, therefore depicting secondary structural changes with an increase in secondary structure percentage suggesting that these compounds interact with PfHsp70-1.

Oxidative testicular injury: effect of L-leucine on redox, cholinergic and purinergic dysfunctions, and dysregulated metabolic pathways.

The antioxidant and anti-proinflammatory activities of L-leucine were investigated on oxidative testicular injury, ex vivo. In vitro analysis revealed L-leucine to be a potent scavenger of free radicals, while inhibiting acetylcholinesterase activity. Oxidative injury was induced in testicular tissues using FeSO4. Treatment with L-leucine led to depletion of oxidative-induced elevated levels of NO, MDA, and myeloperoxidase activity, with concomitant elevation of reduced glutathione and non-protein thiol levels, SOD and catalase activities. L-leucine caused a significant (p < 0.05) alteration of oxidative-elevated acetylcholinesterase and chymotrypsin activities, while concomitantly elevating the activities of ATPase, ENTPDase and 5'-nucleotidase. L-leucine conferred a protective effect against oxidative induced DNA damage. Molecular docking revealed molecular interactions with COX-2, IL-1 beta and iNOS. Treatment with L-leucine led to restoration of oxidative depleted ascorbic acid-2-sulfate, with concomitant depletion of the oxidative induced metabolites: D-4-Hydroxy-2-oxoglutarate, L-cystine, adenosine triphosphate, maleylacetoacetic acid, cholesteryl ester, and 6-Hydroxy flavin adenine dinucleotide. Treatment with L-leucine reactivated glycolysis while concomitantly deactivating oxidative-induced citrate cycle and increasing the impact-fold of purine metabolism pathway. L-leucine was predicted not to be an inhibitor of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, with a predicted LD50 value of 5000 mg/Kg and toxicity class of 5. Additionally, L-leucine showed little or no in vitro cytotoxicity in mammalian cells. These results suggest the therapeutic potentials of L-leucine on oxidative testicular injury, as evident by its ability to attenuate oxidative stress and proinflammation, while stalling cholinergic dysfunction and modulating nucleotide hyrolysis; as well as modulate oxidative dysregulated metabolites and their pathways.

Iso-Mukaadial Acetate from Warburgia salutaris Enhances Glucose Uptake in the L6 Rat Myoblast Cell Line.

Diabetes mellitus (DM) is a chronic metabolic disorder which has become a major risk to the health of humankind, as its global prevalence is increasing rapidly. Currently available treatment options in modern medicine have several adverse effects. Thus, there is an urgent need to develop alternative cost-effective, safe, and active treatments for diabetes. In this regard, medicinal plants provide the best option for new therapeutic remedies desired to be effective and safe. Recently, we focused our attention on drimane sesquiterpenes as potential sources of antimalarial and antidiabetic agents. In this study, iso-mukaadial acetate (Iso) (1), a drimane-type sesquiterpenoid from the ground stem bark of Warburgia salutaris, was investigated for glucose uptake enhancement in the L6 rat myoblast cell line. In vitro assays with L6 skeletal muscle cells were used to test for cytotoxicity, glucose utilisation, and western blot analysis. Additionally, the inhibition of carbohydrate digestive enzymes and 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity were analysed in vitro. The cell viability effect of iso-mukaadial acetate was the highest at 3 µg/mL with a percentage of 98.4. Iso-mukaadial acetate also significantly and dose-dependently increased glucose utilisation up to 215.18% (12.5 µg/mL). The increase in glucose utilisation was accompanied by enhanced 5' adenosine monophosphate-activated protein kinase (AMPK)and protein kinase B (AKT) in dose-dependent manner. Furthermore, iso-mukaadial acetate dose-dependently inhibited the enzymes α-amylase and α-glucosidase. Scavenging activity against DPPH was displayed by iso-mukaadial acetate in a concentration-dependent manner. The findings indicate the apparent therapeutic efficacy of iso-mukaadial acetate isolated from W. salutaris as a potential new antidiabetic agent.