An investigation into the anti-obesity properties of Cyclopia

Jack, Babalwa Unice (2016-12)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH SUMMARY: Polyphenols have a range of health promoting effects against chronic diseases such as obesity and type 2 diabetes mellitus. Previous research in our group showed that Cyclopia species (honeybush), endemic South African plants, display anti-obesity effects. The aim of this study was to investigate the anti-obesity potential of polyphenol-enriched extracts of three Cyclopia spp. (C. subternata, C. intermedia and C. maculata) using bioactivity guided fractionation to facilitate the identification of anti-obesity polyphenols. Aqueous methanol extracts of C. subternata, C. intermedia and C. maculata were prepared and separated into their aqueous and organic fractions, which were tested for their anti-obesity effects in 3T3-L1 adipocytes using the Oil Red O, glycerol release (marker of lipolysis), and triglyceride assays. Cytotoxicity was evaluated using the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays. The anti-obesity properties of the organic fraction of the aqueous methanol extract of C. intermedia were evaluated in obese Leprdb/db mice. Thereafter, the organic fraction was separated into four fractions (CCC F1 to CCC F4) using high performance counter-current chromatography (HPCCC), which were tested for their anti-obesity and cytotoxic effects in 3T3-L1 pre-adipocytes and in mature adipocytes. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and quantitative high performance liquid chromatography diode array detection (HPLC-DAD) were used for determination of phenolic composition. The molecular mechanisms of action of the organic fraction and its CCC fractions were assessed with quantitative real-time PCR and western blot analysis. The polyphenol-enriched extracts of C. subternata, C. intermedia and C. maculata, and their aqueous and organic fractions exhibited differences in phenolic composition and bioactivity. The aqueous fractions of C. maculata and C. subternata, containing lower phenolic content than the organic fractions, decreased lipid content in 3T3-L1 adipocytes compared to their organic counterparts. However, the organic fraction of C. intermedia, with higher phenolic content than its aqueous fraction, decreased lipid content compared to its aqueous fraction in 3T3-L1 adipocytes. In Leprdb/db mice, the organic fraction of C. intermedia decreased body weight gain, without affecting food or water consumption. Further fractionation of the organic fraction of C. intermedia by HPCCC provided four fractions with differences in phenolic composition and anti-obesity effects. None of the extracts or fractions, except for the aqueous C. intermedia and organic C. maculata fractions which decreased ATP content, affected cell viability as measured with the MTT and ATP assays during acute treatment. The highest concentrations of CCC F1 and CCC F3 decreased ATP content during chronic treatment in pre-adipocytes. In 3T3-L1 adipocytes, the organic fraction of C. intermedia and CCC fractions modulated the expression of genes implicated in lipid and energy metabolism, whereas the organic fraction of C. intermedia had no effect on the expression of these genes in Leprdb/db mice. In conclusion, the organic fraction of C. intermedia exhibited anti-obesity properties in vitro and in vivo. A flavanone with anti-obesity potential, neoponcirin, was identified in C. intermedia, the first discovery of this compound in Cyclopia spp. Bioactivity guided fractionation of the organic fraction of C. intermedia resulted in CCC fractions retaining different polyphenols with varying anti-obesity effects. These results demonstrate that the anti-obesity potential of C. intermedia is due to the presence of more than one compound, with different mechanisms of action, or the synergistic effects of more than one compound, may contribute to the bioactivity of C. intermedia.

AFRIKAANSE OPSOMMING: Polifenole het ‘n verskeidenheid van voordelige gesondheids-effekte teen chroniese siekte toestande wat obesiteit en diabetes mellitus tipe 2 insluit. Voorafgaande navorsing in ons groep het aangedui dat die uitsluitlik inheems Suid-Afrikaanse Cyclopia-spesies (heuningbos), obesiteits-teenwerkende effekte toon. Die mikpunt van die huidige studie was om die obesiteits-teenwerkende potensiaal van polifenol-verrykte ekstrakte van drie Cyclopia-spesies (C. subternata, C. intermedia en C. maculata) te ondersoek deur bio-aktiwiteitsgerigte fraksionering te gebruik om obesiteits-teenwerkende polifenole te identifiseer. Waterige metanol ekstrakte van C. subternata, C. intermedia en C. maculata was voorberei en geskei in water-oplosbare en organiese fraksies wat dan getoets was vir moontlike obesiteits-teenwerkende eienskappe in 3T3-L1 vetselle deur gebruik te maak van “Oil Red O”, gliserol-vrystelling (‘n merker vir lipolise) en trigliseried toetse. Seldodende effekte is ondersoek deur die 3-[4,5-Di-metielthiazool-2-iel]-2,5-difenieltetrazolium bromied (MTT) and adenosien trifosfaat (ATP) toetse te gebruik. Die obesiteits-teenwerkende effekte van die organiese fraksie verkry vanuit die waterige metanol ekstrak van C. intermedia is verder ondersoek in Leprdb/db-muise. Hierna is die organiese fraksie opgedeel in vier fraksies (CCC F1 tot CCC F4) deur hoë-werkverrigting teen-vloei chromatografie (HPCCC) te gebruik en dan weer getoets vir obesiteits-teenwerkende en seldodende effekte in 3T3-L1 pre-vetselle en volwasse vetselle. Vloeistof-chromatografie gekoppelde massaspektrometrie (LC-MS/MS) en kwantitatiewe hoë-werkverrigting vloeistof-chromatografie met diode-reeks deteksie (HPLC-DAD) is gebruik vir die bepaling van die fenoliese samestelling. Die molekulêre werkingsmeganismes van die organiese fraksie en die CCC-fraksies is ondersoek deur die kwantitatiewe “intyds” polimerase kettingreaksie en western-klad tegnieke te gebruik. Die polifenol-verrykte ekstrakte van C. subternata, C. intermedia en C. maculata en hul wateroplosbare en organiese fraksies het verskille getoon in fenoliese samestelling asook bio-aktiwiteit. Die wateroplosbare fraksies van C. maculata en C. subternata, met laer fenoliese inhoud as die organiese fraksies, het vet-inhoud in 3T3-L1 vetselle verlaag relatief tot hul ooreenstemmende organiese fraksies. Die organiese fraksie van C. intermedia, met hoër fenoliese inhoud as die wateroplosbare fraksie, het egter die vetinhoud van die 3T3-L1 vetselle verlaag in vergelyking met die ooreenstemmende wateroplosbare fraksie. In Leprdb/db-muise het die organiese fraksie van C. intermedia ‘n verlaging in liggaamsgewigstoename tot gevolg gehad sonder om die voedsel- of water-inname te beïnvloed. Verdere skeiding van die organiese fraksie van C. intermedia met HPCCC het 4 fraksies opgelewer wat elkeen verskil het van die ander in terme van fenoliese samestelling en obesiteits-teenwerkende effekte. Geen van die ekstrakte of fraksies, behalwe die wateroplosbare fraksie van C. intermedia en die organiese fraksie van C. maculata wat ATP-inhoud laat daal het in 3T3-L1 vetselle, het sel-lewensvatbaarheid beïnvloed soos gemeet in MTT- en ATP-toetse na akute behandeling nie. Tydens chroniese behandeling van 3T3-L1 pre-vetselle, het die hoogste konsentrasies van CCC F1 en CCC F3 die ATP-inhoud verlaag. In 3T3-L1 vetselle, het die organiese fraksie van C. intermedia en daaropvolgende CCC-fraksies die uitdrukking van gene betrokke by vet en energie metabolisme gemoduleer terwyl die organiese fraksie van C. intermedia nie die uitdrukking van hierdie gene in Leprdb/db-muise beïnvloed het nie. In samevatting, die organiese fraksie van C. intermedia het obesiteits-teenwerkende eienskappe getoon in vitro en in vivo. ‘n Flavanoon met obesiteits-teenwerkende potensiaal, neo-ponsirien, is geidentifiseer in C. intermedia en is vir die eerste keer gevind in Cyclopia spesies. Bio-aktiwiteitsgerigte fraksionering van die organiese fraksie van C. intermedia het CCC-fraksies gelewer wat verskillende polifenole bevat het en wat verskillende obesiteits-teenwerkende effekte getoon het. Hierdie resultate dui daarop dat die obesiteits-teenwerkende potensiaal van C. intermedia nie toegeken kan word aan ‘n enkele fenoliese verbinding nie, maar dat sinergistiese of kumulatiewe effekte vanaf meer as een obesiteits-teenwerkende verbinding, met verskille in werkingsmeganisme, betrokke mag wees.

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