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Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa
Thesis (PhD)--Stellenbosch University, 2022.
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2022
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| author | Zonyane-Egbichi, Samkele |
| author2 | Makunga, Nokwanda P.
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| author_browse | Makunga, Nokwanda P.
Zonyane-Egbichi, Samkele |
| author_facet | Makunga, Nokwanda P.
Zonyane-Egbichi, Samkele |
| author_sort | Zonyane-Egbichi, Samkele |
| collection | Thesis |
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| description | Thesis (PhD)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125115 |
| institution | Stellenbosch University (South Africa) |
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| last_indexed | 2026-06-10T12:44:46.833Z |
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| spelling | oai:scholar.sun.ac.za:10019.1/125115 Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa Zonyane-Egbichi, Samkele Makunga, Nokwanda P. Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology. Lessertia frutescens Lessertia frutescens (L.) -- Africa, Southern -- Geographical distribution Antioxidants -- Therapeutic use Intraspecific variation Phytochemicals Genetic engineering UCTD Thesis (PhD)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Lessertia frutescens (L.) Goldblatt & J.C. Manning syn. Sutherlandia frutescens (L.) R. BR. is a longstanding botanical remedy that has been used as far back as the 1890s to treat a variety of illnesses. Early records show that medicinal application of L. frutescens originated from the Khoi and Nama people who used decoctions derived from this plant species to treat fevers, wash wounds and for treatment of various ailments. In the present day, L. frutescens is an emerging industrial medicinal plant product and this is mainly due to accumulating scientific evidence that is congruent with claims of its ethnobotanical uses, which include the treatment of internal cancers, diabetes, stress, fevers,wounds, inflammation and stomach complaints. L. frutescens is endemic to southern Africa; growing in Namibia, Botswana, Zimbabwe and South Africa. Within South Africa, L. frutescens has a wide distribution and there is still fragmented information with regards to its variation in the chemical profiles of specimens growing in different geographic locations. Chemotypic variation has bearing on the quality, safety, efficacy and consistency of the medicinal plant preparation. The overarching goal of this dissertation, therefore, was to explore and demonstrate a holistic approach to the quality control assessment of L. frutescens and its medicinal use. It was hypothesized that L. frutescens plants from different geographic origins, within South Africa, would exhibit phytochemical variation. To test this hypothesis, the chemotypic variations in L. frutescens plants growing in different geographic localities were evaluated to generate integrated information on the phytochemical variation of this plant species. To this end, both targeted and non-targeted metabolomics approaches were applied using tandem liquid chromatography mass spectrometry (LC-MS) in combination with chemometric analysis techniques. L. frutescens plants were collected from the seven geographic regions which have not been investigated before, across four provinces of South Africa, namely, the Northern Cape, Western Cape, Eastern Cape and Free State (Chapter 3). The findings of the study showed that plants from different geographic locations had variable sutherlandin contents, thereby, separating plants from the seven geographic localities into three distinct chemical clusters as demonstrated in the principal component analysis (PCA). The extent of phytochemical variation demonstrated by the principal components was 34.47% and 23.68% for PC 1 and for PC 2, respectively. Furthermore, chemical differentiation was also observed for chemometric analysis based on sutherlandiosides. The plants from Northern Cape (Victoria West) had a unique chemical profile that was mainly characterized by the presence of sutherlandioside C. While another set of plants from Northern Cape (Colesburg), Eastern Cape (Burgersdorp) and Free State (Zastron) had similar chemical profiles which contained the major triterpene glycoside, sutherlandioside B. Again, the plants from Western Cape (Gansbaai and Pearly Beach) separated from other plants from other geographic localities and formed their own cluster in the PCA due to the presence of a compound with a mass-to-charge ratio of 737.4104. The PCA demonstrated a total variance of the principal components at 64.40% for PC 1 and 11.30% for PC 2, for the sutherlandiosides. Furthermore, even plants in close proximity to each other (from the same geographic localities) demonstrated phytochemical variation when chemical profiles were further analyzed with orthogonal partial least squares discriminant analysis (OPLS-DA) that reduces the dimensions of the dataset. Using the non- targeted approach, the clustering pattern of L. frutescens plants was similar to that of sutherlandioside-based chemotypic variation. In Chapter 3, the assessment of chemotypic variation was followed by toxicity analysis of pooled samples of L. frutescens. The ethanol and aqueous extracts were screened for their effect on the hatching rate of zebrafish embryos and mortality of larvae at different concentrations that ranged from 5 to 300 µg.ml⁻¹. L. frutescens extracts affected the hatching rate of zebrafish embryos whereby the hatching rate decreased with the increasing concentration of the extracts. Likewise, the mortality rate of larvae increased with the increasing concentration of extracts. However, the aqueous extracts were relatively less toxic compared to ethanol extracts. Thereafter, the implication of phytochemical variation in the biological activity of L. frutescens was assessed in Chapter 4 of the dissertation, wherein it is hypothesized that L. frutescens specimens that exhibit phytochemical variation will likewise show discrepancies in their biological activities. To test this hypothesis, the solvent extracts of L. frutescens, collected from various localities, were comparatively screened for antioxidant capacity using 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging method and ferric reducing antioxidant power (FRAP). The solvent extracts of S. frutescens were further screened for anticancer activity using CellTiter-Glo Viability Assay onDLD-1 colon cancer cell lines. The antioxidant capacity of L. frutescens specimens from different geographic origins differed significantly (p < 0.05) with IC50 values ranging from 3.171 to 7.707 µg.ml⁻¹ and 14.397 to 16.232 mM Trolox equivalent per grams of dry extract for DPPH and FRAP assays, respectively. For anticancer activity, all solvent extracts variably induced cytotoxicity (p < 0.05) on DLD-1 colon cancer cells. It was then expected that if L. frutescens from different geographic origins exhibit phytochemical variation, then these specimens are genetically differentiated. To test this hypothesis, L. frutescens plants were assessed for genetic differentiation, as described in Chapter 5, to see whether there is genetic basis for chemotypic variation observed. The intraspecific genetic variation in L. frutescens, was assessed using the universal chloroplast non-coding DNA fragment that is known to evolve faster than coding regions. Thus, the trnH-trnK chloroplast DNA intergenic spacer was chosen and used to obtain information regarding the genetic diversity and structure of the study populations of S. frutescens. Chloroplast DNA consensus sequences from a total of 33 L. frutescens specimens had a total length of 954 base pairs wherein six haplotypes and 11 polymorphic sites were identified. The total nucleotide diversity (Pi) was 0.00162 and the haplotype diversity (Hd) was 0.716. The majority of genetic variation was partitioned among the plants from different localities at 66% compared to genetic diversity of specimens collected from the same geographic origins which only accounted for 34% of total genetic variation. The limited genetic diversity within plants from the same geographic origin was likely caused by a low gene flow value of 1.2 Nm that was recorded. High genetic differentiation, on the other hand, might explain chemotypic variation that has been recorded in L. frutescens specimens from different localities. However, the phylogenetic clustering pattern which consisted of three main clusters was different from those patterns related to chemotypic variation which also consisted of three main clusters. Overall, for the first time a combination of two ‘omics’ approaches, metabolomics and genomics in addition to chosen bioassays were used as tools to explore biogeographical chemotypes of of L. frutescens as a means to provide a more holistic approach to the study of population-driven quality control aspects in this important medicinal species. Similar to most botanical remedies used in traditional medicine, there is no standardization of the preparation methods and consumption of L. frutescens as yet. Therefore, this study has made significant novel contributions that will ultimately assist in the quest for commercial quality assurance in one of the most important medicinal plants used in South Africa. This study ultimately has bearing for safety, efficacy and predictable therapeutic action of this plant species. The new knowledge that has been generated is one of the steps towards establishment of protocol for the quality assurance and standardization of botanical remedies, that would promote integration and recognition of traditional medicinal plants into formal national healthcare systems. AFRIKAANSE OPSOMMING: Lessertia frutescens (L.) Goldblatt & J.C. Manning syn. Sutherlandia frutescens (L.) R. BR. is vanaf die 1890s al gebruik as `n botaniese geneesmiddel vir verskeie siektes. Die mediese gebruike van L. frutescens se oorsprong is van die Khoi en Nama stamme wat decoctions (gestoomde material) van die specie gebruik het om koors, wonde skoon temaak en as behandeling vir verskillende kwale. Tans word L. frutescens gesien as `n opkommende industrial medisinale plant produk, dit is hoofsaaklik as gevolg van die bykommende wetenskaplike bewyse wat saam met sy volkbotaniese gebruike ooreen stem, dit sluit in die behandeling van interne kanker, diabetes, stres, koors, wonde, inflamasie en maag probleme. L. frutescens is endemies aan suidelike Afrika; waar dit in Namibia, Botswana, Zimbabwe en Suid Afrika groei. Binne die grense van Suid Afria het L. frutescens `n wye verspruiding and daar is steeds gefragmenteerde inligting in verband met die variasie van die chemise profile van individeë wat groei in verskillende areas. Chemotipiese variase het `n impak op die kwaliteit, veiligheid, matigheid en konsitensie van die mediese plant preperaat. Die algehele doel van die proef, was om `n holistiese benadering te ontdek en te demonstreer vir die gehalte beheer te assesseer vir L. frutescens en die medisinale gebruike. Dit was gehipotiseer dat L. frutescens plante van verskillende geografiese oorsprong, binne Suid Afrika, sal fitochemesie variasie toon. Om die hipotese te toets was L. frutescens plante met chemotipiese variasie beoordeel om geïntegreerde inligting van die fitochemise variasie van die plant specie te genereer. Met die hulp van beide geteikende en nie-geteikende metabelomika beginsels was toegepas met behulp van vloeibare kromatografie massa spektrometrie (LC-MS) in kombinasie met chemometriese analiese. L. frutescens plante was verkry vanaf sewe geografiese areas wat nog nie gebestudeer was nie, dit sluit vier van die provinsies van Suid Afrika, naamlik die Noord Kaap, Wes Kaap, Oos Kaap en die Vrystaat (Hoofstuk 3). Die bevindinge van die studie het gewys dat plante van die verskillende geografiese areas verskillende sutherlandin konsentrasies bevat, daardeur, het gelei tot die afskorting van die plante van die sewe geografiese areas in drie duidelike chemise groepe soos aangedui deur die hoof komponent analiese (PCA). Die omvang van fitochemiese variasie gewys deur die hoof kompotnente was 34.47% en 23.68% vir PC 1 en PC 2 respektief. Nogtans, chemise differensasie was ook opgelet in die chemometriese analiese gebaseer op sutherlandiosides. Die plante van die Noord-Kaap (Victora Wes) het `n unieke chemise profile wat hoofsaaklik gekenmerk word deur die teenwoordigheid van sutherlandioside C. Terwyl `n ander stel plante van die Noord-Kaap (Kolesberg), Oos – Kaap (Burgersdorp) en Vrystaat (Zastron) soortgelyke chemise profiele wat hoofsaaklik die hoof triterpene glycoside, sutherlandioside B bevat. Die plante van die Wes-Kaap (Gansbaai en Pearly Beach) het verskil van ander plante en ander geografiese gebiede deur hul eie groep in die PCA te vorm weens `n molekûle met `n gewig tot lading verhouding van 737.4104. Die PCA het gedemonstreer totale varaise van die hoof component by 64.40% vir PC 1 en 11.30% vir PC 2, vir die sutherlandiosides. Boonop, plante wat selfs naby mekaar is (van dieselfde geografiese gebied) het fitochemiese variasie getoon wanneer geanaliseer was met orthogonal partial least squares discriminant analysis (OPLS-DA) wat die omvang van die datastel verklein. Met die hulp van `n nie-geteikende benadering, het die groepering van L. frutescens plante geneig na sutherlandioside-gebaseerde chemotipiese variasie. In Hoofstuk 3, the beraaming van chemotipiese variase was gevold deur `n giftigheid analiese van gekombineerde monsters van L. frutescens. Die ethanol en wateragtige ekstrak was gesif vir hul effek op die uitbroei van zebrafis embrios en die sterftes van larva by verskillende konsentrasies wat gestrek het van 5 tot 300 µg.ml⁻¹. L. frutescens ekstrak het die uitbroei van zebrafish embrios beinvloed waar dit die uitbroei tyd verlaag het met die verhooging van ekstrak konsentrasie. Soortgelyk het die sterfte tempo van larva verhoog met die verhooging van ekstrak konsentrasie. Nogtans, was die wateragtige ekstrak minder giftig in vergelyking met ethanol ekstrakie. Vervolgens, die implikasie van fitochemiese variasie van die biologiese aktiviteit van L. frutescens was gebestudeer in Hoofstuk 4 van die proef, waar dit gehipotiseer was dat L. frutescens monsters wat fitochemiese variasie getoon het sou teenstrydighede vertoon het in hul biologiese aktiviteit. Om die hipotese te toets, was oplosing ekstrasies van L. frutescens wat verkry was van verskillende areas, deeglik gesif vir antioksidant aktiviteit met behulp van 2,2-diphenyl- 1-picrylhydrazyl (DPPH•) radikaal opsoek metode en ferric reduseerende antioksidant krag (FRAP). Die oplossing ekstrak van S. frutescens was verder gesif vir anti-kanker aktiviteit met behulp van CellTiter-Glo Viability Assay op DLD-1 kolon kanker sel lyne. Die antioksidany kapasiteit van L. frutescens monserts van die verskillende areas het beduidend verskil (p < 0.05) met IC50 waardes wat strek van 3.171 tot 7.707 µg.ml⁻¹ en 14.397 tot 16.232 mM Trolox gelykstaande per gram droeë ekstrak vir DPPH en FRAP assays. Vir anti-kanker aktivteit, het alle oplossing ekstraksies verskillende sitotoksiteit (p < 0.05) op DLD-1 kolon kanker selle geinduseer. Daar was verwag dat as L. frutescens van verskillende geografiese oorsprong verskillende fitochemiese variasie toon, dan sal die monsters geneties defferansieer van mekaar. Om die hipotese te toets, was L. frutescens plante gebestudeer vir hul genetiese differensiasie, soos beskryf in Hoofstuk 5, om te sien of daar `n genetiese fondasie is vir die opgelete chemotipiese variasie. Die interspesefieke genetiese variase in L. frutescens was bepaal met behulp van die universele cloroplas nie-kodering DNS fragment wat bekend is om vinniger te evolueer as ander koderings areas. Dus, was die trnH-trnK chlorplas DNS intergeniese spasieerder gekies en gebruik om inligting te verky van die genetiese variasie en struktuur van die bestudeerde bevolking van S. frutescens. Chloroplas DNS ooreenstemende reeks van `n total van 33 L. frutescens monsters het `n totale lengte van 954 basis paar waarin ses haplotipes en 11 polimorfiese areas geidentifiseer. Die totale nukleotied diversiteit (Pi) was 0.00162 en haplotipe diversiteit (Hd) was 0.716. Die meeste genetiese variasie was verdeel tussen plante van verskillende areas by 66% in vergelying met die genetiese variasie van die monsters wat verkry was van die selfde geografiese oorsprong het slegs 34% van die globale genetiese variasie beskryf. Die lae genetiese variasie tussen plante van dieselfde geografiese oorsprong was moontlik veroorsaak deur `n lae geen vloei waarde van 1.2 Nm wat genoteer was. Hoë genetiese deffirensasie, mag dalk die chemotipiese variasie wat genoteer was in L. frutescens monsters van verskillende areas verduidelik. Nogtans, die filogenetiese groepering patroon wat bestaande was uit drie hoof groepe was verskillend van die patrone wat opgetel was vir die chemotipiese variasie wat ook uit drie groepe bestaan het. Algeheel, hier is vir die eerste keer `n gekombineerde benadering van twee ‘omika’, metabolomika en genomika met geselekteerde bioassays gebruik as gereedskap om die biogeografiese chemotipes van L. frutescens te bestudeer as `n metode om `n meer holistiese benadering vir die bestudering van populasie gedrewe gehalte beheer aspekte vir hierdie belangrike medisinale plant specie Soortgelyk aan meeste botaniese geneesmiddels wat gebruik word in tradisionele medesyne, is daar geen standardiseering van die voorbereidings metode en verbruik van L. frutescens tot en met hede nie. Dus, hierdie studie het `n beduidende nuwe insig gelewer wat aan die einde sal help in die ondersoek van kommersieel gehalte besuur vir een van die belangrikste medisinale plante wat gebruik word in Suid Afrika. Hierdie studie op die ou einde het `n impak of veiligheid, matigheid en voorspelbare terapetiese aksie van die plant specie. Die nuwe kennes wat hier gegenereer was is een stap nader om `n protokol in plek te stel vir gehalte beheer en die standardiseering van botaniese geneesmiddels, wat sal help met die integrasie en erkenning van tradisionele medisinale plante in `n formele nationaale gesondheid sisteme. Doctorate 2022-03-04T11:02:38Z 2022-04-29T12:54:37Z 2022-03-04T11:02:38Z 2022-03 Thesis http://hdl.handle.net/10019.1/125115 en_ZA Stellenbosch University xxiv, 163 pages : illustrations (some color), maps application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Lessertia frutescens Lessertia frutescens (L.) -- Africa, Southern -- Geographical distribution Antioxidants -- Therapeutic use Intraspecific variation Phytochemicals Genetic engineering UCTD Zonyane-Egbichi, Samkele Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa |
| title | Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa |
| title_full | Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa |
| title_fullStr | Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa |
| title_full_unstemmed | Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa |
| title_short | Metabolomic analysis, genetic analysis and biological activities of Lessertia Frutescens (L.) Goldblatt & J.C. Manning populations in South Africa |
| title_sort | metabolomic analysis genetic analysis and biological activities of lessertia frutescens l goldblatt j c manning populations in south africa |
| topic | Lessertia frutescens Lessertia frutescens (L.) -- Africa, Southern -- Geographical distribution Antioxidants -- Therapeutic use Intraspecific variation Phytochemicals Genetic engineering UCTD |
| url | http://hdl.handle.net/10019.1/125115 |
| work_keys_str_mv | AT zonyaneegbichisamkele metabolomicanalysisgeneticanalysisandbiologicalactivitiesoflessertiafrutescenslgoldblattjcmanningpopulationsinsouthafrica |