Possible involvement of sialidase and sialyltransferase activities in a stage-dependent recycling of sialic acid in some organs of type 1 and type 2 diabetic rats.

Background: Type 1 (T1D) and type 2 (T2D) diabetes lead to an aberrant metabolism of sialoglycoconjugates and elevated free serum sialic acid (FSSA) level. The present study evaluated sialidase and sialyltranferase activities in serum and some organs relevant to diabetes at early and late stages of T1D and T2D. Methods: Sialic acid level with sialidase and sialyltransferase activities were monitored in the serum, liver, pancreas, skeletal muscle and kidney of diabetic animals at early and late stages of the diseases. Results: The FSSA and activity of sialidase in the serum were significantly increased at late stage of both T1D and T2D while sialic acid level in the liver was significantly decreased in the early and late stages of T1D and T2D, respectively. Furthermore, the activity of sialidase was significantly elevated in most of the diabetes-relevant organs while the activity of sialyltransferase remained largely unchanged. A multiple regression analysis revealed the contribution of the liver to the FSSA while pancreas and kidney contributed to the activity of sialidase in the serum. Conclusions: We concluded that the release of hepatic sialic acid in addition to pancreatic and renal sialidase might (in)directly contribute to the increased FSSA during both types of diabetes mellitus.

Phytochemical evaluation of Ziziphus mucronata and Xysmalobium undulutum towards the discovery and development of anti-malarial drugs.

BACKGROUND: The development of resistance by Plasmodium falciparum is a burdening hazard that continues to undermine the strides made to alleviate malaria. As such, there is an increasing need to find new alternative strategies. This study evaluated and validated 2 medicinal plants used in traditional medicine to treat malaria. METHODS: Inspired by their ethnobotanical reputation of being effective against malaria, Ziziphus mucronata and Xysmalobium undulutum were collected and sequentially extracted using hexane (HEX), ethyl acetate (ETA), Dichloromethane (DCM) and methanol (MTL). The resulting crude extracts were screened for their anti-malarial and cytotoxic potential using the parasite lactate dehydrogenase (pLDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. This was followed by isolating the active compounds from the DCM extract of Z. mucronata using silica gel chromatography and structural elucidation using spectroscopic techniques (NMR: 1H, 12C, and DEPT). The active compounds were then targeted against P. falciparum heat shock protein 70-1 (PfHsp70-1) using Autodock Vina, followed by in vitro validation assays using ultraviolet-visible (UV-VIS) spectroscopy and the malate dehydrogenase (MDH) chaperone activity assay. RESULTS: The extracts except those of methanol displayed anti-malarial potential with varying IC50 values, Z. mucronata HEX (11.69 ± 3.84 µg/mL), ETA (7.25 ± 1.41 µg/mL), DCM (5.49 ± 0.03 µg/mL), and X. undulutum HEX (4.9 ± 0.037 µg/mL), ETA (17.46 ± 0.024 µg/mL) and DCM (19.27 ± 0.492 µg/mL). The extracts exhibited minimal cytotoxicity except for the ETA and DCM of Z. mucronata with CC50 values of 10.96 and 10.01 µg/mL, respectively. Isolation and structural characterization of the active compounds from the DCM extracts revealed that betulinic acid (19.95 ± 1.53 µg/mL) and lupeol (7.56 ± 2.03 µg/mL) were responsible for the anti-malarial activity and had no considerable cytotoxicity (CC50 > µg/mL). Molecular docking suggested strong binding between PfHsp70-1, betulinic acid (- 6.8 kcal/mol), and lupeol (- 6.9 kcal/mol). Meanwhile, the in vitro validation assays revealed the disruption of the protein structural elements and chaperone function. CONCLUSION: This study proves that X undulutum and Z. mucronata have anti-malarial potential and that betulinic acid and lupeol are responsible for the activity seen on Z. mucronata. They also make a case for guided purification of new phytochemicals in the other extracts and support the notion of considering medicinal plants to discover new anti-malarials.

Ursolic acid acetate and iso-mukaadial acetate bind to Plasmodium falciparum Hsp90, abrogating its chaperone function in vitro.

Plasmodium falciparum is the most lethal malaria parasite. Increasing incidences of drug resistance of P. falciparum have prompted the need for discovering new and effective antimalarial compounds with an alternative mode of action. Heat shock protein 90 (PfHsp90) facilitates protein folding and is a promising antimalarial drug target. We have previously reported that iso-mukaadial acetate (IMA) and ursolic acid acetate (UAA) exhibit antimalarial activity. We investigated the abilities of IMA and UAA to bind PfHsp90 by molecular docking and dynamics simulations. The in silico predictions were validated by biochemical assays conducted on recombinant PfHsp90. The interaction between the ligands and PfHsp90 was evaluated using ultraviolet-visible spectroscopy (UV-vis), Fourier transform infrared (FTIR), and surface plasmon resonance (SPR) analysis. The results obtained by docking calculations and MD dynamics simulation predicted that UAA and IMA preferentially bound to PfHsp90 via the N-terminal domain, with UAA binding more stable than IMA. UV-vis-based data suggest that PfHsp90 harbors buried aromatic amino acids, which were exposed in the presence of either IMA or UAA. In addition, data obtained using FTIR suggested that IMA and UAA destabilized the secondary structure of PfHsp90. Of the two compounds, UAA bound to PfHsp90 within the micromolar range based on surface plasmon resonance (SPR)-based binding assay. Furthermore, both compounds disrupted the holdase chaperone function of PfHsp90 as the chaperone failed to suppress heat-induced aggregation of the model proteins, malate dehydrogenase (MDH), luciferase, and citrate synthase in vitro. In addition, both compounds lowered the ATPase activity of PfHsp90. The molecular dynamics simulation analysis indicated that the docked complexes were mostly stable for 100 ns, validating the data obtained through the biochemical assays. Altogether, this study expands the repository of antiplasmodial compounds that have PfHsp90 among their possible targets.

Therapeutic efficacy of ß-sitosterol treatment on Trypanosoma congolense infection, anemia development, and trans-sialidase (TconTS1) gene expression.

Background: African animal trypanosomiasis hinders sustainable livestock productivity in sub-Saharan Africa. About 17 million infected cattle are treated with trypanocides annually but most of the drugs are associated with drawbacks, necessitating the search for a promising chemotherapeutic agent. Objectives: In this study, the effects of ß-sitosterol on Trypanosoma congolense infection were investigated along with its effect on the trans-sialidase gene expressions. Results: Oral treatment with ß-sitosterol at 15 and 30 mg/kg body weight (BW) for 14 days significantly (p < 0.05) reduced parasitemia and ameliorated the parasite-induced anemia. Also, the parasite-induced increase in serum urea level and renal histopathological damage scores in addition to renal hypertrophy was significantly (p < 0.05) reverted following treatment with 30 mg/kg BW ß-sitosterol. The compound also significantly (p < 0.05) down-regulated the expression of TconTS1 but not TconTS2, TconTS3, and TconTS4. Correlation analysis between free serum sialic acid with the TconTS1 and TconTS2 gene variants revealed negative correlations in the ß-sitosterol-treated groups although they were non-significant (p > 0.05) in the group treated with 15 mg/kg BW ß-sitosterol. Similarly, a non-significant negative (p > 0.05) correlation between the biomolecule and the TconTS3 and TconTS4 gene variants was observed in the ß-sitosterol-treated groups while positive correlations were observed in the infected untreated control group. Conclusion: The observed effect of ß-sitosterol on T. congolense infection could make the compound a possible template for the design of novel trypanocides.

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.

Evaluating Iso-Mukaadial Acetate and Ursolic Acid Acetate as Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase Inhibitors.

To date, Plasmodium falciparum is one of the most lethal strains of the malaria parasite. P. falciparum lacks the required enzymes to create its own purines via the de novo pathway, thereby making Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPT) a crucial enzyme in the malaria life cycle. Recently, studies have described iso-mukaadial acetate and ursolic acid acetate as promising antimalarials. However, the mode of action is still unknown, thus, the current study sought to investigate the selective inhibitory and binding actions of iso-mukaadial acetate and ursolic acid acetate against recombinant PfHGXPT using in-silico and experimental approaches. Recombinant PfHGXPT protein was expressed using E. coli BL21 cells and homogeneously purified by affinity chromatography. Experimentally, iso-mukaadial acetate and ursolic acid acetate, respectively, demonstrated direct inhibitory activity towards PfHGXPT in a dose-dependent manner. The binding affinity of iso-mukaadial acetate and ursolic acid acetate on the PfHGXPT dissociation constant (KD), where it was found that 0.0833 µM and 2.8396 µM, respectively, are indicative of strong binding. The mode of action for the observed antimalarial activity was further established by a molecular docking study. The molecular docking and dynamics simulations show specific interactions and high affinity within the binding pocket of Plasmodium falciparum and human hypoxanthine-guanine phosphoribosyl transferases. The predicted in silico absorption, distribution, metabolism and excretion/toxicity (ADME/T) properties predicted that the iso-mukaadial acetate ligand may follow the criteria for orally active drugs. The theoretical calculation derived from ADME, molecular docking and dynamics provide in-depth information into the structural basis, specific bonding and non-bonding interactions governing the inhibition of malarial. Taken together, these findings provide a basis for the recommendation of iso-mukaadial acetate and ursolic acid acetate as high-affinity ligands and drug candidates against PfHGXPT.

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.

Isolation, characterization, and biological evaluation of a potent anti-malarial drimane sesquiterpene from Warburgia salutaris stem bark.

BACKGROUND: Malaria continues to be a global burden as the efficacy of most commercial anti-malarial drugs has been compromised by the evolution of parasite resistance. With the urgent need created for the development of alternative and more efficient anti-malarial drugs, this study focused on the evaluation of anti-malarial agents of the Warburgia salutaris stem bark. METHODS: The stem bark was extracted with dichloromethane followed by silica gel column chromatography that led to the isolation of iso-mukaadial acetate, a drimanoid sesquiterpene. This compound was characterized by spectroscopic analysis (1H NMR, 13C NMR, IR and MS), and its structure was confirmed by X-ray crystallography. In vitro anti-plasmodial activity was investigated using a chloroquine sensitive NF54 Plasmodium falciparum strain. Cytotoxicity was measured using the MTT assay on HEK239 and HEPG2 cell lines. Chloroquine-sensitive Plasmodium berghei was used to infect Sprague-Dawley rats for in vivo studies. The W. salutaris crude extract and iso-mukaadial acetate were administered orally at 0.5; 1.5, 2.5 and 5 mg/kg, chloroquine was used as the reference drug. Determination of percentage parasitaemia, haematological parameters, and rat body weights was done throughout the experimental study period. RESULTS: The crude extract and iso-mukaadial acetate showed very good activity on the inhibition of parasite growth (IC50 0.01 ± 0.30 µg/ml) and (IC50 0.44 ± 0.10 µg/ml), respectively, with iso-mukaadial acetate having cytotoxicity activity of 36.7 ± 0.8 and 119.2 ± 8.8 (µg/ml) on HEK293 and HEPG2 cells, respectively. The crude extract and iso-mukaadial acetate reduced percentage parasitaemia in a dose-dependent manner in comparison to the control. There were no significant differences in the haematological parameters in all the experimental groups in comparison to control group. This study proves that W. salutaris contains components (including iso-mukaadial acetate) that exhibit anti-malarial activity. This study scientifically validates the use of this plant in folk medicine. CONCLUSIONS: This study proves that Warburgia salutaris contains components (including iso-mukaadial acetate) that exhibit anti-malarial activity and scientifically validates the use of this plant in folk medicine.

Anti-plasmodial activity of some Zulu medicinal plants and of some triterpenes isolated from them.

Mimusops caffra E. Mey. ex A.DC and Mimusops obtusifolia Lam (both members of the Sapotaceae family), and Hypoxis colchicifolia Bak (family Hypoxidaceae) are used by traditional healers in Zululand to manage malaria. Anti-plasmodial investigation of the crude extracts and some triterpenes isolated from the plants showed activity against a chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10). Among the crude extracts the leaves of M. caffra exhibited the highest activity, with an IC50 of 2.14 µg/mL. The pentacyclic tritepenoid ursolic acid (1), isolated from the leaves of M. caffra was the most active compound (IC50 6.8 µg/mL) as compared to taraxerol (2) and sawamilletin (3) isolated from the stem bark of M. obtusifolia (IC50 > 100). Chemical modification of the ursolic acid (1) to 3ß-acetylursolic acid (4) greatly enhanced its anti-plasmodial activity. Compound 4 reduced parasitaemia against Plasmodium berghei by 94.01% in in vivo studies in mice. The cytotoxicity of 3ß-acetylursolic acid (IC50) to two human cell lines (HEK293 and HepG2) was 366.00 µg/mL and 566.09 µg/mL, respectively. The results validate the use of these plants in folk medicine.