Development of a gastroretentive anti-diabetic nutraceutical incorporating polyphenol-enriched fractions of Cyclopia genistoides

Miller, Neil (2020-04)

Thesis (PhD)--Stellenbosch University, 2020.

Thesis

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.

AFRIKAANSE OPSOMMING: Ekstrakte van heuningbos (Cyclopia genistoides) met ‘n hoë inhoud van die geglikosileerde xantone, mangiferien (1) en isomangiferien (2), en die bensofenone, 3-β-D-glukopiranosieliriflofenoon (3) en 3-β-D- glukopiranosiel-4-O-β-D-glukopiranosieliriflofenoon (4), inhibeer α-glukosidase (AG), ‘n belangrike spysverteringsensiem en behandelingsteiken vir postprandiale hiperglukemie geassosieer met tipe 2 diabetes. Ultrafiltreerde groen C. genistoides ekstrak het gedien as beginmateriaal vir die ontwikkeling van ‘n geoptimiseerde produksieprotokol vir xantoon- en bensofenoon-verrykte fraksies (XVFs en BVFs) d.m.v. makroporeuse adsorberende hars chromatografie. Variasie in fenoliese inhoud tussen verskillende produksielotte van die roumateriaal het gemanifesteer as variasie in die samestelling en mate van teikenverbindingverryking in XVFs en BVFs. Die in vitro AG inhiberingsvermoë van C. genistoides fenole, ekstrak, XVF en BVF, gekombineer met akarbose, ‘n kommersiële AG inhibeerder (AGI), is ondersoek d.m.v. die kombinasie indeks. Die rang-orde van die AGIs se potensie was as volg: akarbose (IC50 = 44.3 µM) > 1 (102.2 µM) > 2 (119.8 µM) > 3 (237.5 µM) > 4 (299.4 µM). Die potensie van die ekstrak en fraksies was sterk verwant aan hul xantooninhoud. XVFs, met xantooninhoud tussen 22.3 en 48.1 g/100 g, is geproduseer van tien verskillende produksielotte van plantmateriaal, en getoets teen ‘n vaste konsentrasie (160 µg/mL), met resulterende ensieminhibering van 63–72%. Die BVFs, met bensofenooninhoud tussen 11.4 en 21.7 g/100 g, het ensieminhibering van 26–34% bewerkstellig by dieselfde konsentrasie. Daar was ‘n swak lineêre korrelasie (R² < 0.43) tussen die teikenverbindinginhoud van fraksies en hul AG inhiberingspotensie. Sinergistiese inhibering van AG by > 50% effektiwiteitsvlakke is waargeneem vir alle kombinasies van akarbose met fraksies (XVFs, BVFs) of teikenverbindings (1–4). Kombinasies van akarbose met 1 en 2 het die hoogste teoretiese in vitro akarbose dosisverlaging (> sesvoudig) bewerkstellig by alle effektiwiteitsvlakke. XVFs het beter akarbose dosisverlaging (≈ viervoudig by 50% effektiwiteitsvlak) bewerkstellig as BVFs, wat dui op die potensiaal van XVFs as aanvuller tot akarbose. In ‘n daaropvolgende in vivo orale sukrose toleransie toets in normale en diabetiese Wistar rotte, het XVF (enkele orale dosis van 300 mg/kg liggaamsgewig) nie die postprandiale bloedglukose noemenswaardig verlaag nie, en ook nie ‘n verbeterde effek in kombinasie met akarbose (5 mg/kg liggaamsgewig) bewerkstellig nie. Die geskiktheid van C. genistoides fenole as potensiële nie-toksiese aktiewe farmaseutiese bestanddele (AFBs) is bevestig d.m.v. ‘n lewer-sel model, waarin geen sitotoksiteit waargeneem is na akute of chroniese blootstelling nie. Ex vivo intestinale transportstudies met varkderm het aangedui dat die teikenverbindings (1–4) swak geabsorbeer word, wat hul geskiktheid as AFBs met ‘n intestinale teiken bevestig, asook die lae risiko vir sistemiese toksisiteit herbeklemtoon. XVF en BVF is geïnkorporeer (alleen en gekombineerd) in ‘n nie-bruisende gastro-retentiewe afleweringsisteem met laedigtheid stireen-divinielbenseen ko-polimeer as die dryfagent. Die tablette het in ‘n in vitro medium (0.1 N HCl) vir minstens 8 h gedryf, en die AFBs is vrygestel d.m.v. ‘n diffusie-gebaseerde proses wat met die wiskundige model van Weibull beskryf is (R² > 0.99). Afbreking van AFBs gedurende opberging onder nadelige toestande (12 weke teen 40 °C) het eerste-orde reaksiekinetika gevolg met ‘n chemiese stabiliteitsrangorde van 4 > 1 > 2 > 3.

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