An exploratory investigation into the physicochemical, antioxidant and cellular effects of a selection of honey samples from the Southern African region

The unique floral biodiversity of Southern Africa would be reflected in the phenolic acid and flavonoid composition as well as the antioxidant activity of honeys from this region. In this exploratory investigation the total polyphenolic (TPC) and flavonoid (TFC) content, antioxidant activity as well as the cellular protective effects of a selection of honeys collected in this region was evaluated. Thirteen honey samples representative of the Western Cape (WCa, WCb and WCc), Eastern Cape (ECa, ECb and ECc), South East Mozambique (SEMa, SEMb and SEMc) and Agricultural: A-E (Eucalyptus) (A-E1 and A-E2), A-L (Litchi) and A-O (Orange) were collected. These samples were subjected to physicochemical analysis, the antioxidant content (TPC and TFC) and both enzymatic (catalase activity) and non-enzymatic activity, using the 2,2-diphenyl-2-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays was determined. From the DPPH, TEAC and ORAC data the Relative Antioxidant Capacity Index (RACI) was calculated. To determine whether high antioxidant activity translates into significant cellular protection, biological and cellular assays were undertaken. Using the pBR322 plasmid assay and the erythrocyte haemolysis assay the ability of honeys to protect against 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) oxidative damage was evaluated. Further evaluation was undertaken in the SC-1 fibroblast cell line and the physiologically more relevant Caco-2 cell line. Toxicity and antioxidant effects were evaluated in the SC-1 cell line while antioxidant effects were only evaluated in the Caco-2 cell line. The long-term mitogenic and toxic effects were determined in the SC-1 cell line using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Neutral Red (NR) and Crystal Violet (CV) assays. Short term, total- and intracellular antioxidant effects were determined in both cell lines using the dichlorofluorescein diacetate assay (DCFH-DA) assay. For all cellular experiments honey at concentrations of 0.01% and 1% were used. The physiochemical properties of the honeys evaluated fulfilled the regulatory standards compiled in the Codex Alimentarius (CODEX STAN12-1981 revision 2001). The results were as follows: SEMb had the highest TPC (167.96 mg GAE/100g) and TFC (51.60 mg CE/100g) while A-E2 had the highest catalase (38.48 µmol H2O2/g) activity. RACI revealed that WCb had the highest antioxidant activity.SEMc showed the highest protection of plasmid DNA against oxidative-induced strand breaks while SEMa showed the highest protection of erythrocytes against AAPH-induced haemolysis. Although correlations were found between antioxidant content and antioxidant activity assays, no correlation was found these parameters and the biological assays. For the long-term cytotoxicity assay, AAPH showed significant cytoxicity at 0.78mM, 1.56mM and 0.28mM when measured using the MTT, NR and CV assays, respectively. Some honeys 4/13 and 3/13 showed a mitogenic effect at a concentration of 0.01% and 1% respectively. Toxic effects, were observed for 1/13 and 8/13 at 0.01% and 1% honey respectively. Toxicity after 72 h exposure varied from 10-30% (CV assay). The same concetrations of honey was used to determine the short-term, 2h, antioxidant effects in both the SC-1 and Caco-2 cell lines. No oxidative effect was found for all honeys at these concentrations. For the DCFH-DA assay using the SC-1 cell line at 1%, 12/13 and 7/13 honeys showed total and intracellular protection respectively. The highest extracellular protection was for SEMa (% Protection (%P) = 95) and SEMb (%P = 93). Intracellular protection was the highest for SEMc (%P = 21) and A-L (%P = 20). At 0.01%, 7/13 and 8/13 honeys exhibited total and intracellular protection, respectively. For both the highest protection was found for SEMc (%P = 43, total and %P = 30, intracellular). For the Caco-2 cell line at 1%, 11/13 and 4/13 showed total and intracellular protection, respectively. Of these the highest extracellular protection was for SEMb (% Protection (%P) = 90). Intracellular protection was the highest for ECa (%P = 28) and WCc (%P = 26). At 0.01%, 4/13 and 8/13 honeys showed total and intracellular protection respectively. The highest extracellular protection was found for SEMc (%P = 62) and intracellular protection was ECc (%P = 28). The SC-1 cell line was found to be the most sensitive to the antioxidant effects of honey compared to the Caco-2 cell line. The honeys SEMa, SEMb and SEMc showed protection against oxidative damage in both cell lines. In conclusion, the antioxidant activity of honeys from Southern Africa is of a high quality. The WC, SEM and EC honeys showed the highest antioxidant effects and could provide health benefits against diseases associated with oxidative stress as indicated by these results. CopyrightDissertation (MSc)--University of Pretoria, 2011.Anatomyunrestricte

Stability, morphology, and effects of in vitro digestion on the antioxidant properties of polyphenol inclusion complexes with -cyclodextrin

DATA AVAILABILTY STATEMENT : Some data is available as supplementary material and the rest under the UPSpace institutional repository, URI: http://hdl.handle.net/2263/84453 (date dissertation was available online 11 March 2022).SUPPLEMENTARY MATERIAL : FIGURE S1: Enhanced product ions scan mass spectrum of CD inclusion complexes with (a) CAT, (b) GA, and (c) EGCG. CD—beta cyclodextrin, CAT—catechin, GA—gallic acid, EGCG—epigallocatechin gallate; FIGURE S2: Inhibition of AGEs formation by non-digested (ND) and following simple (SD) and complex (CD) digestion of nonencapsulated and encapsulated CAT/GA, CAT/EGCG, and GA/EGCG combinations evaluated with the (a) BSA-MGO and (b) BSA-FRU models at 100 M for each polyphenol in each sample. The data is represented as the mean SEM of at least five experiments performed in duplicates. ND—nondigested, SD—simple digestion, CD—complex digestion, AGEs—advanced glycation end-products, BSA—bovine serum albumin, MGO—methylglyoxal, FRU—fructose, CAT—catechin, GA—gallic acid, EGCG—epigallocatechin gallate. The * and + represent significant (p < 0.05) differences between non-encapsulated and encapsulated ND compared with the respective SD or CD samples. The denotes significant (p < 0.05) differences between the non-encapsulated and encapsulated for each treatment; TABLE S1: Polyphenol–polyphenol interactions on antioxidant activity (ORAC assay ( M TE)); TABLE S2: Polyphenol–polyphenol interactions on antiglycation activity (AGEs assay (%)); TABLE S3: Polyphenol–polyphenol interactions on cellular antioxidant activity (DCFH-DA assay (%)).Polyphenols are inversely associated with the incidence of chronic diseases, but therapeutic use is limited by poor stability and bioaccessibility. Encapsulation has been shown to overcome some of these limitations. A selection of polyphenols (catechin, gallic acid, and epigallocatechin gallate) and their combinations were encapsulated in beta-cyclodextrin (CD). Encapsulation was characterized and the thermal and storage stability was evaluated using the 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) assay. The samples were then subjected to in vitro digestion using a simple digestion (SD) model (gastric and duodenal phases) and a more complex digestion (CD) model (oral, gastric, and duodenal phases). Thereafter, the chemical (oxygen radical absorbance capacity assay) and cellular (dichlorofluorescein diacetate assay in Caco-2 cells) antioxidant and antiglycation (advanced glycation end-products assay) activities were determined. Inclusion complexes formed at a 1:1 molar ratio with a high encapsulation yield and efficiency. Encapsulation altered the morphology of the samples, increased the thermal stability of some and the storage stability of all samples. Encapsulation maintained the antioxidant activity of all samples and significantly improved the antiglycation and cellular antioxidant activities of some polyphenols following SD. In conclusion, the formed inclusion complexes of CD with polyphenols had greater storage stability, without altering the beneficial cellular effects of the polyphenols.The National Research Foundation South Africa and the APC was funded by the University of Pretoria, Faculty of Health Sciences Library and the University of Pretoria, Faculty of Health Sciences.https://www.mdpi.com/journal/moleculesam2023AnatomyPharmacolog

Chemokines as possible therapeutic targets in metastatic melanoma

DATA AVAILABILITY STATEMENT : Data sharing not applicable to this article as no datasets were generated or analysed during the current study.BACKGROUND : Cutaneous melanoma is a relentless form of cancer which continues to rise in incidence. Currently, cutaneous melanoma is the leading cause of skin cancer-related mortality, which can mainly be attributed to its metastatic potential. The activation of chemokine axes is a major contributor to melanoma metastasis through its involvement in promoting tumour cell migration, proliferation, survival, and adhesion. This review will focus on the role of chemokines in melanoma and possible therapeutic strategies to alter chemokine activation and subsequently inhibit the activation of signalling cascades that may promote metastasis. METHODS : A literature review was conducted to evaluate chemokines as possible therapeutic targets in metastatic melanoma. RESULTS : The crosstalk between signalling pathways and immune responses in the melanoma microenvironment resembles a complex and dynamic system. Therefore, the involvement of governing chemokine axes in the promotion of cutaneous and metastatic melanoma demands a proper understanding of the tumour microenvironment in order to identify possible targets and develop appropriate treatments against melanoma. CONCLUSION : Even though chemokine axes are regarded as promising therapeutic targets, it has become increasingly evident that chemokines can play a critical role in both tumour inhibition and promotion. The inhibition of chemokine axes to inhibit signalling cascades in target cells that regulate metastasis should, therefore, be carefully approached.The National Research Foundation (NRF); the School of Medicine Research Committee (RESCOM) and the University of Pretoria.http://www.wileyonlinelibrary.com/journal/cam4hj2023AnatomyPhysiolog

The tryptophan–kynurenine pathway in immunomodulation and cancer metastasis

DATA AVAILABILITY STATEMENT : Data sharing not applicable to this article as no datasets were generated or analysed during the current study.INTRODUCTION : The activation of the kynurenine pathway in cancer progression and metastasis through immunomodulatory pathways has drawn attention to the potential for kynurenine pathway inhibition. The activation of the kynurenine pathway, which results in the production of kynurenine metabolites through the degradation of tryptophan, promotes the development of intrinsically malignant properties in cancer cells while facilitating tumour immune escape. In addition, kynurenine metabolites act as biologically active substances to promote cancer development and metastasis. METHODS : A literature review was conducted to investigate the role of the tryptophan-kynurenine pathway in immunomodulation and cancer metastasis. RESULTS : Evidence suggests that several enzymes and metabolites implicated in the kynurenine pathway are overexpressed in various cancers. As such, the tryptophan pathway represents a promising target for cancer treatment. However, downstream signalling pathways, including aryl hydrocarbon receptor activation, have previously induced diverse biological effects in various malignancies, which resulted in either the promotion or the inhibition of metastasis. CONCLUSION : As a result, a thorough investigation of the kynurenine pathway and its regulatory mechanisms is necessary in order to properly comprehend the effects of kynurenine pathway activation involved in cancer development and metastasis.The University of Pretoria, School of Medicine Research Committee (RESCOM) and National Research Foundation (NRF).http://wileyonlinelibrary.com/journal/cam4hj2023AnatomyPhysiolog

Antioxidant and anti-inflammatory properties of Ilex guayusa tea preparations : a comparison to Camellia sinensis teas

Ilex guayusa tea preparations are now commercially available as Runa tea. Little is known regarding the antioxidant and anti-inflammatory bioactivities of this tea. The I. guayusa teas had a total polyphenolic content between 54.39 and 67.23 mg GAE/g dry mass and peroxyl radical scavenging capacities between 1773.41 and 2019 µmol TE/g dry mass, nearly half of that for the Camellia sinensis teas. The I. guayusa teas afforded 60-80% protection from oxidative stress in the Caco-2 cellular antioxidant assay, comparable to the C. sinensis teas. The anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 cells of I. guayusa teas was similarly comparable to the C. sinensis teas with nitric oxide production reduced by 10-30%. Major compounds identified by mass spectrometry were the phenolic mono- and dicaffeoylquinic acid derivatives. I. guayusa teas are a good source of dietary phenolic compounds with cellular antioxidant and anti-inflammatory properties.http://pubs.rsc.org/en/journals/journalissues/fo#!recentarticles&adv2018-12-13hj2017AnatomyBiochemistr

The dipeptidyl peptidase IV inhibitory activity and multifunctional antidiabetic properties of SQSPA : structure – activity relationship evaluated with alanine scanning

Type 2 diabetes is a multifactorial disease and drugs with multifunctional properties are required. The peptide, SQSPA, was reported to be a potent and gastrointestinally stable α-glucosidase inhibitory peptide. In this study, the structure-activity relationship of this peptide was studied using alanine scanning. Four analogs; AQSPA, SASPA, SQAPA and SQSAA were designed and investigated for multifunctional antidiabetic effects. Molecular docking studies on human dipeptidyl peptidase-IV (DPP-IV) suggested that the binding affinities were in the order; AQSPA>SASPA>SQSPA>SQSAA>SQAPA while for in vitro DPP-IV inhibitory activity, it was SQSPA>SQSAA>AQSPA>SASPA>SQAPA. Enzyme kinetic studies revealed that the peptides are uncompetitive inhibitors with the exception of SQSAA and SQSPA. In 3T3-L1 differentiated adipocytes, SASPA was the only analog that significantly (p < 0.05) reduced and prevented lipid accumulation and did not induce cytotoxicity to differentiated 3T3-L1 cells. All peptides, especially SASPA scavenged methylglyoxal and peroxyl radicals thereby preventing advanced glycosylated end products formation and oxidative stress. The nitric oxide scavenging activity of all peptides was comparable to IPI and glutathione. Findings indicate that the amide side chain of Q2 is probably the most critical functional group for modulating the multifunctional antidiabetic effects of SQSPA while SASPA has been identified, as a novel peptide with enhanced multifunctional antidiabetic activity.The National Research Foundation of South Africa and the University of Pretoria.http://www.elsevier.com/locate/ijbiomac2021-10-01hj2020AnatomyBiochemistryGeneticsMicrobiology and Plant Patholog

How methylglyoxal kills bacteria : an ultrastructural study

Antibacterial activity of honey is due to the presence of methylglyoxal (MGO), H2O2, bee defensin as well as polyphenols. High MGO levels in manuka honey are the main source of antibacterial activity. Manuka honey has been reported to reduce the swarming and swimming motility of Pseudomonas aeruginosa due to de-flagellation. Due to the complexity of honey it is unknown if this effect is directly due to MGO. In this ultrastructural investigation the effects of MGO on the morphology of bacteria and specifically the structure of fimbriae and flagella were investigated. MGO effectively inhibited Gram positive (Bacillus subtilis; MIC 0.8 mM and Staphylococcus aureus; MIC 1.2 mM) and Gram negative (P. aeruginosa; MIC 1.0 mM and Escherichia coli; MIC 1.2 mM) bacteria growth. The ultrastructural effects of 0.5, 1.0 and 2 mM MGO on B. substilis and E. coli morphology was then evaluated. At 0.5 mM MGO, bacteria structure was unaltered. For both bacteria at 1 mM MGO fewer fimbriae were present and the flagella were less or absent. Identified structures appeared stunted and fragile. At 2 mM MGO fimbriae and flagella were absent while the bacteria were rounded with shrinkage and loss of membrane integrity. Antibacterial MGO causes alterations in the structure of bacterial fimbriae and flagella which would limit bacteria adherence and motility.http://www.tandfonline.com/loi/iusp202017-03-31hb2016AnatomyBiochemistr

Whole blood ultrastructural alterations by mercury, nickel and manganese alone and in combination : an ex vivo investigation

The distribution of metals across the environment is increasingly becoming a major concern as they not only pollute the environment but also pose a danger to humans and animals. Human exposure to heavy metals often occurs as a combination of metals the synergistic effects of which can be more toxic than a single metal. The aim of this study was to investigate the effects that the metals mercury (Hg), nickel (Ni) and manganese (Mn) alone and in combination have on erythrocyte morphology and other components of the coagulation system using the haemolysis assay, scanning electron microscopy (SEM), and confocal laser scanning microscopy. Human blood was exposed to the heavy metals ex vivo, and percentage haemolysis was determined. Ultrastructural analysis of erythrocytes, platelets and fibrin networks was performed using SEM. Analysis of phosphatidylserine (PS) flip-flop was determined using confocal laser scanning microscopy. At the highest concentration of 10,000× the World Health Organization safety limit, all the metals caused haemolysis. The results showed that the exposure of erythrocytes to Hg alone and in combination with other metals displayed more haemolysis compared to Ni and Mn alone and in combination. Components of the coagulation system showed ultrastructural changes, including the formation of echinocytes and the activation of platelets with all single metals as well as the combinations. Confocal laser scanning microscopy analysis showed the presence of PS on the outer surface of the echinocytes that were exposed to metals alone and in combination. It can, therefore, be concluded that these heavy metals have a negative impact on erythrocytes and the coagulation system.https://journals.sagepub.com/home/tihhj2022AnatomyPhysiolog

The effects of kynurenine metabolites on cell proliferation and morphology in melanoma and neuroblastoma cell lines

Research Development Programme (RDP) (UP)UCD Granthttps://drive.google.com/file/d/1juIb6_CBLaV_p1W3lCCoiy6DKY4CoWJB/view?usp=sharinghttps://drive.google.com/drive/folders/15c8nNl2iUi3wTrFeIUNHHMx9ctqb2gcc?usp=sharinghttps://drive.google.com/drive/folders/1fwqlMm1hRp4TyUwXiAXdvF6yDWyPHgcb?usp=sharin

Generation of reactive oxygen species in relevant cell lines as a bio-indicator of oxidative effects caused by acid mine water

Reactive oxygen species (ROS) production and resultant oxidative stress (OS) has been implicated as a pathway of toxicity in animal species exposed to pollutants. The gills of aquatic animals and the liver and kidneys of mammalian species are specific cellular sites of toxicity. Oxidative effects of acid mine drainage effluent (following passive and active treatment) impacting a natural stream were assessed using selected cell lines. Levels of pollutants such as heavy metals in acid mine drainage (AMD) effluent can be quantified following treatment, but it is unknown whether this is associated with equivalent reduction in toxicity. ROS production by AMD untreated (U) and after treatment (T) was quantified in a fish gill cell line (RTgill-W1) and in two mammalian cell lines (C3A human liver and Vero monkey kidney). ROS production was determined using the oxidant sensitive fluorogenic probe, 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) following exposure to U and T, AMD water. Treatment of AMD water caused reduction in levels of Al, Zn, Fe, Si and Mn while levels of Cr, Cu, Ar and Hg remained unchanged. A dose-dependent increase in ROS production was observed for U and T. ROS formation decreased from 14% to 4.5%, 16.4% to 7.2% and 25.3% to 17.7% in the RTgill-W1, C3A, and Vero cell lines exposed to 100% AMD water, U and T. The presence of Mn and/or other ions in treated water and subsequent ROS formation indicates that water could still be toxic to cells and requires further processing. The DCFH-DA assay in several cell lines can be used to rapidly bio-monitor quality of AMD water related to formation of ROS and subsequent cellular effects. However, cut-off levels for cellular toxicity must be established to ensure safety of this water for aquatic animals and for animal and human consumption.OTI is grateful to the Schlumberger Stichting Fund, The Netherlands for a fellowship. The National Research Foundation and the Department of Paraclinical Sciences (University of Pretoria) are acknowledged for research funding [Project number: V027-12].http://www.wrc.org.zaam2017AnatomyParaclinical Science