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Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush)
Thesis (PhDFoodSc)--Stellenbosch University, 2019.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2019
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| _version_ | 1867613933399965696 |
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| access_status_str | Open Access |
| author | Walters, Nico Albertus |
| author2 | De Beer, Dalene |
| author_browse | De Beer, Dalene Walters, Nico Albertus |
| author_facet | De Beer, Dalene Walters, Nico Albertus |
| author_sort | Walters, Nico Albertus |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhDFoodSc)--Stellenbosch University, 2019. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/106926 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:01Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/106926 Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush) Walters, Nico Albertus De Beer, Dalene Joubert, Elizabeth Williams, Paul James Stellenbosch University. Faculty of Agrisciences. Dept. of Food Science. Honeybush (Cyclopia) -- Phenology High performance liquid chromatography Countercurrent chromatography Natural deep eutectic solvents Cyclopia pubescens -- Composition Phenolic compounds UCTD Thesis (PhDFoodSc)--Stellenbosch University, 2019. ENGLISH ABSTRACT: Cyclopia pubescens Eckl. & Zeyh, endemic to South Africa, is under threat of extinction. This threat can be negated through commercial cultivation if utilised as honeybush tea. Lack of knowledge of the phenolic composition of this Cyclopia species is a hurdle in the development of value-added products such as nutraceuticals, providing the incentive for a comprehensive investigation of the phenolic profile of C. pubescens. A reversed-phase (RP) high-performance liquid chromatography-diode-array detection (HPLC-DAD) method using a biphenyl column was developed and validated. Eight phenolic compounds, representing major compounds (3-β-D-glucopyranosyl-4-O-β-D-glucopyranosyliriflophenone, 3-β-D- glucopyranosylmaclurin, 3-β-D-glucopyranosyliriflophenone, mangiferin, isomangiferin, vicenin-2, narirutin and hesperidin), were identified and a further six compounds tentatively identified by comparison of their retention times, UV–Vis spectra and high-resolution mass spectrometric (HR-MS) characteristics with those of authentic reference standards and literature, respectively. The predominant accumulation of the compounds in the leaves or stems of C. pubescens was determined for a small number of available genotypes (n = 17), affording some indication of variation between plants in the field genebank of the Agricultural Research Council. For further elucidation of the phenolic profile, the focus fell on minor phenolic compounds. Increased separation power was required and therefore an analytical comprehensive two-dimensional (2D) separation method utilising normal-phase (NP) high- performance countercurrent chromatography-DAD (HPCCC) as the first dimension and RP ultra(U)HPLC-DAD as the second dimension. High loading capacity and selectivity of HPCCC and high selectivity and resolving power of UHPLC resulted in ca 81% orthogonality. A total of 32 compounds were (tentatively) identified by comparing their UV– Vis and HR-MS characteristics to those of reference standards and literature. The HPCCC method was subsequently upscaled to semi-preparative mode and eight phenolic compounds were isolated in adequate quantities for unambiguous structural elucidation using 2D nuclear magnetic resonance, gas chromatography-MS analysis after acid- catalysed hydrolysis of the glycoside, derivatisation and determination of optical activity. (2R)-5-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]eriodictyol, (2S)-5-O-[α-L- rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]eriodictyol and (2S)-5-O-[α-L-rhamnopyranosyl- (1→2)-β-D-glucopyranosyl]-5,7,3ʹ,4ʹ-tetrahydroxyflavan have not been previously reported in literature. The five other compounds include the two flavanones, (2S)-5-O-[α-L- rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]naringenin and R-neo-eriocitrin, the two phenolic acids, 3-O-α-L-arabinopyranosyl-3,4-dihydroxybenzoic acid and 4-O-β-D-glucopyranosyl-cis-4-hydroxycinnamic acid, and an anisole, 4-(4ʹ-O-β-D-glucopyranosyl-3ʹ- methoxyphenyl)-2-butanone. Natural deep eutectic solvents (NADES) (n = 7) were evaluated to improve extraction efficiency and obtain a more representative phenolic profile of C. pubescens. Hot water and 40% EtOH, commonly used for extraction of Cyclopia plant material, represented the benchmark solvents. Stereoisomers were selectively extracted by the different NADES solvents. Application of an on-line HPLC-ABTS assay showed that these stereoisomers also differed in Trolox equivalent antioxidant capacity. Glutamic acid based NADES (n = 2) provided superior extraction efficiency, but low eutectic stability made these solvents unsuitable for high-throughput extraction; therefore, the most suitable NADES for extracting the phenolic compounds of C. pubescens was choline chloride:proline (CcP) (1:3, molar ratio). The optimal extraction temperature for NADES, tested over the range 40 to 90 °C, was 70 °C instead of 93 °C for water when using a 20 min extraction time. The best extraction solvent for phenolic compounds remains 40% EtOH, but NADES can be used to selectively extract phenolic compounds. AFRIKAANSE OPSOMMING: Cyclopia pubescens Eckl. & Zeyh, endemies aan Suid-Afrika, is ‘n bedreigde plant spesie, maar kommersiële verbouiing kan uitwissing voorkom. Die huidige gebrek aan inligting rakende die fenoliese samestelling van die plant kniehalter ontwikkeling van produkte met toegevoegde waarde soos nutraseutiese produkte, wat die aansporing vir ‘n omvattende studie rakende die fenoliese samestelling van C. pubescens geskep het. ‘n Skeidingsmetode bestaande uit omgekeerde fase (RP) hoë-werkverrigting vloeistof chromatografie (HPLC) met ultraviolet-fotodiode deteksie (DAD gepaardgaande met ‘n bi-feniel kolom is ontwikkel en gevalideer. Agt van die prominente fenoliese verbindings (3-β-D-glukopiranosiel-4-O-β-D-glukopiranosieliriflofenoon, 3-β-D- glukopiranosielmaklurien, 3-β-D- glukopiranosieliriflofenoon, mangiferien, isomangiferien, visenien-2, narirutien en hesperidien) is geϊdentifiseer en ‘n verdere ses verbindings is tentatief geϊdentifiseer deur onderskeidelik gebruik te maak van retensietye, ultra-violet spektra en hoë resolusie massa spektrometrie (HR-MS) saam met, onderskeidelik, dié van outentieke fenoliese standaarde en literatuur data. Analise van die blare en stokgedeeltes van 17 genotipes beskikbaar in veld genebank van die Landbounavorsing Raad het getoon dat die fenoliese verbindings hoofsaaklik in die blare akkummuleer, so wel as ‘n aanduiding van kwantitatiewe verskille tussen genotipes gegee. ‘n Omvattende, af-lyn, twee-dimensionele (2D) skeidingsmetode is ontwikkel om in- diepte kwalitatiewe inligting oor die fenoliese samestelling te bekom. ‘n Normale fase (NP) hoë werkverrigting vloeistofvloeistof chromatografie (HPCCC) metode is as eerste dimensie skeiding ontwikkel met RP ultra (U)HPLC as die tweede dimensie. Die hoë selektiwiteit en laaikapasiteit van HPCCC gepaardgaande met die hoë selektiwiteit en resolusie van UHPLC het ‘n hoë graad van ortogonaliteit (~81%) tot gevolg gehad. ‘n Totaal van 32 verbindings is (tentatief) geϊdentifiseer deur gebruik te maak van standaarde, literatuur data en gepaardgaande ultra-violet en HR-MS data. Die HPCCC metode is opgeskaal na ‘n semi- preparatiewe skaal waarna agt fenoliese verbindings in genoegsame hoeveelhede geïsoleer is vir identifikasie deur gebruik te maak van 2D kern magnetiese resonansie, gas chromatografie-MS analiese na hidroliese en derivatisering van die suikers, en spesifieke rotasie van verbindings. (2R)-5-O-[α-L-rhamnopiranosiel-(1→2)-β-D-glukopiranosiel]- eriodiktiol, (2S)-5-O-[α-L-rhamnopiranosiel-(1→2)-β-D-glukopiranosiel]eriodiktiol en (2S)-5- O-[α-L-rhamnopiranosiel-(1→2)-β-D-glukopiranosiel]-5,7,3ʹ,4ʹ-tetrahydroxyflavaan is vir die eerste keer geϊdentifiseer. Vyf ander verbindings, naamlik twee flavanone, (2S)-5-O-[α-L- rhamnopiranosiel-(1→2)-β-D-glukopiranosiel]naringenien en R-neo-eriositrien, twee fenoliese sure, 3-O-α-L-arabinopiranosiel-3,4-dihydroksibensoësuur en 4-O-β-D- glukopiranosiel-cis-4-hidroksiekaneelsuur, en ‘n anisool, 4-(4ʹ-O-β-D-glukopiranosiel-3ʹ- metoksifeniel)-2-butanoon is ook geϊdentifiseer. Gebruik van natuurlike diep eutektiese oplosmiddels (NADES) (n = 7) om ‘n meer verteenwoordige fenoliese ekstrak met verhoogte ekstraksie doeltreffendheid te verkry, is ook ondersoek. Warm water en 40% EtOH wat as algemene ekstraksie middels vir Cyclopia plant materiaal gebruik word, is as maatstaf gebruik. NADES kon selektief R en S stereoisomere onttrek uit die plant material. ‘n Aan-lyn HPLC-ABTS toets het aangedui dat die stereoisomere verskillende Trolox ekwivalent anti-oksidant kapasiteite het. Met die oog op ekstraksie doeltreffendheid was NADES gebaseer op glutamiensuur (n = 2) die beste ekstraksie middel, maar hierdie NADES was eutekties onstabiel en nie geskik vir ‘n hoë deurset toepassing nie. Gevolglik is cholienchloried:prolien (CcP) (1:3, molêre verhouding) gekies as die mees gepaste NADES vir die ekstraksie van fenoliese verbindings van C. pubescens. Ekstraksie by 70 °C is geidentifiseer as die optimale ekstraksie temperatuur vir CcP, gegewe die reeks, 40 – 90 °C. Die beste ekstraksie middel vir fenoliese verbindings bly steeds 40% EtOH, maar NADES toon potensiaal vir selektiewe ekstraksie van fenoliese subgroepe. Doctoral 2019-11-04T11:23:32Z 2019-12-11T06:39:17Z 2021-04-30T03:00:09Z 2019-12 Thesis http://hdl.handle.net/10019.1/106926 en_ZA Stellenbosch University xv, 236 pages : illustrations (some color), maps application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Honeybush (Cyclopia) -- Phenology High performance liquid chromatography Countercurrent chromatography Natural deep eutectic solvents Cyclopia pubescens -- Composition Phenolic compounds UCTD Walters, Nico Albertus Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush) |
| title | Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush) |
| title_full | Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush) |
| title_fullStr | Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush) |
| title_full_unstemmed | Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush) |
| title_short | Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush) |
| title_sort | comprehensive characterisation of the phenolic composition of cyclopia pubescens honeybush |
| topic | Honeybush (Cyclopia) -- Phenology High performance liquid chromatography Countercurrent chromatography Natural deep eutectic solvents Cyclopia pubescens -- Composition Phenolic compounds UCTD |
| url | http://hdl.handle.net/10019.1/106926 |
| work_keys_str_mv | AT waltersnicoalbertus comprehensivecharacterisationofthephenoliccompositionofcyclopiapubescenshoneybush |