Browsing by Author "Miller, Neil"

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    Development of a gastroretentive anti-diabetic nutraceutical incorporating polyphenol-enriched fractions of Cyclopia genistoides
    (Stellenbosch : Stellenbosch University, 2020-04) Miller, Neil; Joubert, Elizabeth; Manley, Marena; Malherbe, Christiaan Johannes; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Extracts of honeybush (Cyclopia genistoides) containing glycosylated xanthones, mangiferin (1) and isomangiferin (2), and benzophenones, 3-β-D-glucopyranosyliriflophenone (3) and 3-β-D-glucopyranosyl-4- O-β-D-glucopyranosyliriflophenone (4) inhibit α-glucosidase (AG), a key digestive enzyme and treatment target for postprandial hyperglycaemia associated with type 2 diabetes. Ultrafiltered green C. genistoides extract served as the starting material for the development of an optimised production protocol for xanthone- and benzophenone-enriched fractions (XEFs and BEFs) by macroporous adsorbent resin chromatography. Inter-batch variation in the phenolic content of the raw material manifested as variation in the composition and degree of enrichment of target compounds in XEFs and BEFs. The in vitro AG inhibitory effects of C. genistoides phenolics, extract, XEF and BEF, combined with the commercial AG inhibitor (AGI), acarbose, were investigated using the combination index. The single-compound AGIs demonstrated potency in the descending order: acarbose (IC50 = 44.3 µM) > 1 (102.2 µM) > 2 (119.8 µM) > 3 (237.5 µM) > 4 (299.4 µM). Potency of the extract and fractions was strongly linked to their xanthone content. XEFs (xanthone content = 22.3–48.1 g/100 g) were produced using ten different batches of plant material and tested at a fixed concentration (160 µg/mL), achieving 63 to 72% enzyme inhibition. BEFs (benzophenone content = 11.4– 21.7 g/100 g) achieved enzyme inhibition of 26 to 34%. There was a weak linear correlation (R² < 0.43) between the target compound content of the fractions and their AG inhibition potency. Synergistic AG inhibition at > 50% effect levels was observed for all combinations of acarbose with fractions (XEFs, BEFs) or target compounds (1–4). Combinations of acarbose with 1 and 2 gave the highest theoretical in vitro acarbose dose reduction (> six-fold) across all effect levels. XEFs showed greater theoretical acarbose dose reduction (≈ four-fold at 50% inhibition) than BEFs, demonstrating the potential of XEFs as a supplement to acarbose. In a subsequent in vivo oral sucrose tolerance test in normal and diabetic Wistar rats, XEF (single orally administered dose of 300 mg/kg body weight) did not result in significantly lowered postprandial blood glucose or in an improved effect in combination with acarbose (5 mg/kg body weight). The suitability of C. genistoides phenolics as non-toxic active pharmaceutical ingredients (APIs) was confirmed in a liver cell model, which indicated no cytotoxicity following acute or chronic exposure. Ex vivo intestinal transport studies using porcine jejenum showed that the target compounds (1–4) are poorly absorbed, confirming their suitability as APIs aimed at an intestinal target, and re-emphasising the low risk of systemic toxicity. XEF and BEF were subsequently incorporated (alone and combined) in a non-effervescent gastroretentive tablet formulation containing low-density styrene-divinylbenzene co-polymer as floating agent. The tablets floated in an in vitro medium (0.1 N HCl) for at least 8 h and released APIs through a diffusion-based process, described by the Weibull model (R² > 0.99). API degradation during storage under adverse conditions (12 weeks at 40 °C) followed first order reaction kinetics with the order of compound stability: 4 > 1 > 2 > 3.
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    Green rooibos neutraceutical : optimisation of hot water extraction and spray-drying by quality-by-design methodology
    (Stellenbosch : Stellenbosch University, 2016-12) Miller, Neil; Joubert, E.; De Beer, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Unfermented Aspalathus linearis, otherwise known as green rooibos (GR), contains high levels of aspalathin, a potent C-glucosyl dihydrochalcone antioxidant with antidiabetic bioactivity, unique to rooibos. Inherent variation in the phenolic composition of rooibos is likely to cause significant variability in the aspalathin content of different GR production batches and thus also the batch-to-batch quality of a nutraceutical green rooibos extract (GRE). The aim of this study was to optimise hot water extraction and spray-drying for the production of a shelf-stable GRE. A quality-by-design (QbD) approach was applied, entailing a preliminary risk assessment step, one-factor-at-a-time analysis, and analyses according to a central composite design (CCD) to determine the effects of process parameters on responses. Response surface methodology (RSM) was applied to identify suitable control spaces, i.e. ranges of process input factors in which optimal responses, i.e. product quality, could be expected. Significant variation in aspalathin content of different GR production batches (n = 47; 2.5–4.5%) was demonstrated. The CCD for extraction included three independent variables: extraction time (10–40 min), extraction temperature (41–93 °C) and water-to-plant material ratio (6.6:1–23.4:1; v.m-1). Prediction models and response surfaces for extract yield (EY; g.100 g-1 plant material), aspalathin extraction efficiency (Asp_EE; g.100g-1 in plant material) and aspalathin content (g.100 g-1 soluble solids) were generated. Verification of the prediction models showed good predictive ability for EY and Asp_EE. Multi-response optimisation was applied to identify levels of the independent variables which would maximise EY and Asp_EE. Optimal conditions were identified based on these results, along with considerations of cost-efficiency and practicality: extraction time, 29–31 min; extraction temperature, 90–95 °C and water-to-plant material ratio, 9:1–11:1 (v.m-1). Validation of the optimal extraction conditions using the 47 commercial GR production batches, (aspalathin content >2.5%) showed that at least 15% EY and 8% aspalathin content in the extract could be achieved. Standardisation of the maximum particle size by sieving out of large particles could potentially improve the overall process efficiency. The CCD for spray-drying included three independent variables: inlet air temperature (150–220 °C), feed concentration (5–35%) and feed flow rate (0.12–0.64 L.h-1). Powder yield (g powder recovered per 100 g solids in feed) was the only response for which a statistically significant prediction model was generated. Optimal spray-drying conditions (inlet air temperature of 210–230 °C, feed concentration of 34–36% and feed flow rate of 0.62–0.67 L.h-1) were identified and applied in the spray-drying of a pure GRE as well as GRE in a 1:1 mass ratio blend with the carriers, inulin and maltodextrin, respectively. Amorphous powders with low moisture content (<2.2%) and water activity (<0.13) and >89% retention of aspalathin were obtained. The hygroscopic character of the powders was confirmed by moisture sorption analysis, and storage conditions of <25 °C and <40% relative humidity are therefore recommended in order to maintain optimal quality. Inulin improved the flowability and wettability of the powder as compared with maltodextrin. An inulin-GRE formulation was identified as a good candidate for further development as a high-value antidiabetic nutraceutical.
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    In vitro a-glucosidase inhibition by honeybush (Cyclopia genistoides) food ingredient extract-potential for dose reduction of acarbose through synergism
    (Royal Society of Chemistry, 2020-06-29) Miller, Neil; Malherbe, Christiaan J.; Joubert, Elizabeth
    Extracts of Cyclopia species are used as food ingredients. In vitro α-glucosidase (AG) inhibition by ultrafiltered C. genistoides extract, fractions enriched in xanthones (XEF) and benzophenones (BEF), as well as mangiferin, isomangiferin, 3-β-D-glucopyranosyliriflophenone (I3G) and 3-β-D-glucopyranosyl-4-O-β-D-glucopyranosyliriflophenone (IDG) was determined with acarbose as positive control. XEF was more potent than the extract and BEF (IC50 = 43.3, 95.5 and 205.7 μg mL−1, respectively). Compounds demonstrated potency in the descending order: acarbose (IC50 = 44.3 μM) > mangiferin (102.2 μM) > isomangiferin (119.8 μM) > I3G (237.5 μM) > IDG (299.4 μM). The combination index (CI) was used to determine synergism (CI < 0.7) as demonstrated for combinations of acarbose with XEF, BEF or the respective compounds at 50% and 75% effect levels. The greatest potential acarbose dose reductions (>six-fold) across all effect levels were calculated for combinations of acarbose with mangiferin or isomangiferin, explaining the greater acarbose dose reduction potential of XEF vs. BEF. The effect of batch-to-batch variation (n = 10) of raw plant material on AG inhibition was quantified at a fixed concentration (160 μg mL−1). XEFs (xanthone content = 223–481 g kg−1) achieved AG inhibition of 63–72%, whereas BEFs (benzophenone content = 114–251 g kg−1) achieved AG inhibition of 26–34%, with weak linear correlation (R2 < 0.43) between target compound content of the fractions and their achieved AG inhibition. Thus, extract fractions of C. genistoides, enriched in xanthones and benzophenones, show potential in reducing the effective dose of acarbose required to prevent postprandial hyperglycaemia.
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    Model development for predicting in vitro bio-capacity of green rooibos extract based on composition for application as screening tool in quality control
    (Royal Society of Chemistry, 2020-03-09) Viraragavan, Amsha; Hlengwa, Nokulunga; De Beer, Dalene; Riedel, Sylvia; Miller, Neil; Bowles, Sandra; Walczak, Beata; Muller, Christo; Joubert, Elizabeth
    Mounting evidence of the ability of aspalathin to target underlying metabolic dysfunction relevant to the development or progression of obesity and type 2 diabetes created a market for green rooibos extract as a functional food ingredient. Aspalathin is the obvious choice as a chemical marker for extract standardisation and quality control, however, often the concentration of a single constituent of a complex mixture such as a plant extract is not directly related to its bio-capacity, i.e. the level of in vitro bioactivity effected in a cell system at a fixed concentration. Three solvents (hot water and two EtOH–water mixtures), previously shown to produce bioactive green rooibos extracts, were selected for extraction of different batches of rooibos plant material (n = 10). Bio-capacity of the extracts, tested at 10 μg ml−1, was evaluated in terms of glucose uptake by C2C12 and C3A cells and lipid accumulation in 3T3-L1 cells. The different solvents and inter-batch plant variation delivered extracts ranging in aspalathin content from 54.1 to 213.8 g kg−1. The extracts were further characterised in terms of other major flavonoids (n = 10) and an enolic phenylpyruvic acid glucoside, using HPLC-DAD. The 80% EtOH–water extracts, with the highest mean aspalathin content (170.9 g kg−1), had the highest mean bio-capacity in the respective assays. Despite this, no significant (P ≥ 0.05) correlation existed between aspalathin content and bio-capacity, while the orientin, isoorientin and vitexin content correlated moderately (r ≥ 0.487; P < 0.05) with increased glucose uptake by C2C12 cells. Various multivariate analysis methods were then applied with Evolution Program-Partial Least Squares (EP-PLS) resulting in models with the best predictive power. These EP-PLS models, based on all quantified compounds, predicted the bio-capacity of the extracts for the respective cell types with RMSECV values ≤ 11.5, confirming that a complement of compounds, and not aspalathin content alone, is needed to predict the in vitro bio-capacity of green rooibos extracts. Additionally, the composition of hot water infusions of different production batches of green rooibos (n = 29) at ‘cup-of-tea’ equivalence was determined to relate dietary supplementation with the extract to intake in the form of herbal tea.