Metabolomic profiling and micropropagation of Salvia africanalutea L. potent against Fusarium verticillioides

Nkomo, Mpumelelo (2014-02)

Thesis (PhD)--Stellenbosch University, 2014.


ENGLISH ABSTRACT: Salvia africana lutea is one of 26 Sage species indigenous to Southern Africa from a total of 900 worldwide. The genus Salvia belongs to the Lamiaceae family. Labeled a ‘broad spectrum remedy’ S. africana lutea amongst other sage species is medicinally important. Reports are many highlighting its benefits, which include from alleviating coughs and colds to gynaecological complaints. Studies have revealed in vitro antimicrobial, anti-cancer and antioxidant activity. Plant secondary metabolites fundamentally have a strong bearing on the phytochemical activities a plant may possess. Consequently the environment indirectly affects the phytochemical properties as it influences the variation in the plant metabolome via plant-environment interactions. Five S. africana lutea plant populations, within the Western Cape province of South Africa were sampled and chemotypes and bioactivity tested. Four populations were wild growing in protected areas namely; Brackenfell, Koeberg, Silwerstroomstrand and Yzerfontein, while the fifth was a garden growing population from Stellenbosch. Using gas chromatography hyphenated with mass spectrometry (GC-MS), compounds such as monosaccharides, carboxylic acids and fatty acids were detected. Variation of compounds identified with 80% certainty was compared across all populations. Stellenbosch population showed some compounds that were not present in the other four sites. These compounds were namely; propanoic acid, rythronic acid, 2-keto-1-gluconic acid and 1,3-dibromobicyclon, while this population also did not have xylitol that was detected in all the other four populations. To consolidate the GC-MS findings, analysis on the metabolite profiles (utilizing liquid chromatography linked with mass spectrometry (LC-MS) and nuclear magnetic resonance (1H NMR)) was done. Principal component analysis (PCA) was applied to the NMR data. The partial least squaresdiscriminant analysis (PLS-DA) was used to integrate LC-MS and NMR data sets. All statistics were performed with the SIMCA-P+ 12.0 software. By integrating LC-MS and 1H NMR analyses, large chemotype differences leading to samples grouping by site, suggested strong plant-environment interactions as factors influencing metabolite composition. Signals distinguishing the Stellenbosch profile were in the aromatic part of the 1H NMR spectra. Antimicrobial activity was tested against two Fusarium species. Fusarium is a plant pathogenic species that causes large agricultural losses particularly in the maize crop, one of the staple foods in the African continent. Some species also produce mycotoxins in infected crop and lead to a significant increase in the risk factor of cancers when contaminated foods are consumed. Four high-mycotoxin producing strains from two species F. verticillioides (MRC 826 and MRC 8267); F. proliferatum (MRC 7140 and MRC 6809) were utilized in all in vitro antifungal assays in this study. A preliminary assay using dichloromethane: methanol (1:1 v/v) crude plant extracts of the five populations; Stellenbosch, Brackenfell, Koeberg, Silwerstoomstrand and Yzerfontein, from 2009 and 2011. All test samples exhibited good activity as the minimum inhibitory concentrations (MIC) values ranged from 0.031 mg ml-1 to 0.5 mg ml-1, values below the latter are regarded as strong inhibitors. The Stellenbosch extracts were the most active for both 2009 and 2011 collections, with the best activity against F. verticillioides MRC 8267 and MRC 826 at 0.031 mg ml-1. While the least activity, albeit still a strong inhibitor, was observed from the Yzerfontein extracts with an MIC value of 0.5 mg ml-1. Generally comparison between the two years revealed that samples collected in 2011 were more potent than those in 2009, possibly due to prolonged storage that may have resulted in chemical decomposition. As the Stellenbosch population had shown the best activity as well as a relatively different chemical profile, leaves from these plants were then introduced into tissue culture conditions. Leaf explants were placed on solid plant growth regulator (PGR)-free Murashige and Skoog media and that supplemented with hormones in various combinations. (two concentrations of benzyl adenine (BA) utilized individually and in combination with naphthalene acetic acid (NAA) 4.4 and 8.8, while for NAA 0.27, 2.7 and 5.4.) Namely: 4.4 BA, 8.8 BA, 0.27 NAA: 4.4 BA, 2.7 NAA: 4.4 BA, 5.4 NAA: 4.4 BA, 2.7 NAA: 8.8 BA and 5.4 NAA: 8.8 BA. The PGR combinations did not induce shooting nor rooting, only callus on PGR-free MS media. Antifungal activity of the callus extract was in the same range as the whole plant extracts from which the leaf explants were harvested from, showing no ‘loss’ of activity after introduction to tissue culture conditions. Metabolite profiles using LC-MS, however, did reveal qualitative and quantitative differences, though they appear to not have any bearing on the activity. A bioassay-guided fractionation was then conducted on samples collected from Stellenbosch. This led to the identification of carnosol and carnosic acid being involved in the anti-Fusarium activity of S africana-lutea. A combinational study revealed no synergistic activity of the two compounds against four Fusarium test strains, with fractional inhibitory concentration (FIC) values of 1.5 and 3.0. Antifungal activity of carnosol and carnosic acid was observed to be in the same range (strong inhibitor) as was the callus and whole plant extracts. The study showed variation in population chemotypes and identified two compounds that are involved in S. africana-lutea activity against Fusarium species. It also provided a tissue culture system onto which mass production of the two bioactives may be achieved from, in the development of new fungicides.

AFRIKAANSE OPSOMMING: Salvia Africana lutea is een van die 26 Salie spesies wat inheems aan Suid-Afrika is uit ‘n totaal van 900 spesies wêreldwyd. Die genus Salvia hoort tot die Lamiaceae familie. S. africana lutea word geklassifiseer as ‘n “breë spektrum geneesmiddel”, en medisinaal as belangrik geag tussen die ander salie spesies, want volgens verslag word dit gebruik vir die verligting van hoes en verkoues tot selfs ginekologiese ongesteldhede. Definitiewe biologiese aktiwiteit eksperimente het anti-mikrobiese, antikanker en anti-oksidant aktiwiteite aan die lig gebring. Plant sekondêre metaboliete het fundamenteel ‘n baie sterk verband met die fitochemiese aktiwiteite van ‘n plant. Gevolglik affekteer die omgewing indirek die fitochemiese eienskappe, want dit beïnvloed die variasie in die plant metaboloom deur die interaksies van die plant met die omgewing. In vyf streke binne die Wes-Kaap van Suid-Afrika waar S. africana lutea bevolkings voorkom, is steekproewe gedoen en chemotipes en bioaktiwiteit getoets. Vier bevolkings was wild-groeiende bevolkings in beskermde areas, naamlik; Brackenfell, Koeberg, Silwerstroomstrand en Yzerfontein, terwyl die vyfde uit ‘n tuingroeiende bevolking in Stellenbosch geneem is. Deur gas chromatografie gekoppel met massa spektrometrie te gebruik, is primêre samestellings soos monosakkariede, karboksielsure en vetsure gevind. Variasies van samestellings wat met 80% sekerheid geïdentifiseer is, is oorkruis met al die bevolkings vergelyk. Die Stellenbosch bevolking het ‘n paar samestellings geopenbaar wat nie aanwesig was in die ander vier terreine nie. Hierdie samestellings was: propanoësuur, erythroniese suur, 2-keto-1-glukoniese suur en 1,3-dibromobicyclon. Verder het hierdie bevolking geen xylitol gehad nie en dit is in al vier die ander bevolkings gevind. Verdere studies was gedoen met die gebruik van vloeibare chromatografie gekoppel met massa spektrometrie (LC-MS) sowel as kern magnetiese resonansie (1H NMR). Chemiese profiele het hoë variasies getoon, en dus deur te fokus op die aromatiese samestelling streke, het die Stellenbosch terrein duidelik merkbare verskille en punte op die PLS-DA aangetoon. Met die koppeling van NMR data met LC-MS data, is daar gevind dat onderskeidende punte van die NMR PLS-DA wat gegroepeer is met retensie tye die skeiding van die Stellenbosch terrein van ander terreine gedryf het. Dit het onweerlegbaar bewys dat daar variasie binne die vyf bevolkings voorkom en dat Stellenbosch die mees noemenswaardige chemotipe variasie het. Dit blyk uit die anti-mikrobiese eksperimente dat aktiwiteit teen Fusarium heel nuwe belangstelling wek. Fusarium is ‘n plant-patogeniese spesie wat groot landbou verliese veroorsaak veral in die mielie gewasse, een van die stapelvoedsels van die Afrika kontinent. Dit produseer ook mikotoksiene in aangetaste gewasse en hierdie kan lei tot die ontstaan van kankers wanneer besmette voedsel op groot skaal verbruik word. Vier hoë-mikotoksien produserende swamlyne van twee spesies, naamlik F. verticillioides (MRC 826 en MRC 8267) en F. proliferatum (MRC 7140 en MRC 6809) is gebruik in alle in vitro anti-swam ondersoeke in hierdie studie. Die eerste analise het dichloromethan: methanol (1:1 v/v) ongesuiwerde plant ekstrakte bevat van die vyf bevolkings: Stellenbosch, Brackenfell, Koeberg, Silwerstroomstrand en Yzerfontein, geneem gedurende 2009 en 2011. Al hierdie toets monsters het goeie aktiwiteit getoon waar die minimum beperkende konsentrasie (MIC) waardes van 0.031 mg ml-1 tot 0.5 mg ml-1 gevarieer het. Waardes laer as laasgenoemde word beskou as sterk inhibeerders. Die Stellenbosch ekstrakte was die mees aktief vir albei jare hierbo genoem, met die beste aktiwiteit teen F. verticillioides MRC 8267 en MCR 826 by 0.031 mg ml-1. Die minste aktiwiteit (hoewel nog ‘n sterk inhibeerder) waargeneem was van die Yzerfontein ekstrakte, met ‘n MIC waarde van 0.5 mg ml-1. Oor die algemeen het ‘n vergelyking tussen die twee jare aangetoon dat die monsters wat in 2011 versamel is veel sterker was dan dié van 2009, moontlik te wyte aan ‘n verlengde bewaringstyd wat moontlik ‘n chemiese dekomposisie ten gevolge gehad het. Omdat die Stellenbosch bevolking die beste aktiwiteit getoon het sowel as ‘n relatief afwykende chemiese profiel, is blare van hierdie plante toe bekendgestel aan weefselkultuur kondisies. Blaar eksplante is op soliede hormoonvrye Murashige en Skoog media geplaas en dit is aangevul met sintetiese auksien Naftaleen asynsuur (NAA) en sitokien Bensiel adenien (BA) individueel en in verskillende kombinasies. Geen wortels of uitloopsels is waargeneem in al die hormoon kombinasies nie maar in die hormoonvrye media het daar egter Kallus in twintig persent van die eksplante voorgekom. Kallus is toe as subkultuur van hormoonvrye MS media gekweek en saamgevoeg en dichloromethan: methanol (1:1v/v) ekstrakte is getoets teen die volgende Fusarium swamlyne MRC 826; MRC 8267; MRC 7140 en MRC 6809. MIC waardes het sterk inhiberende eienskappe getoon met die laagste waarde as 0.025 mg ml-1 teen drie swamlyne: MRC 1740, MRC 8267 en MRC 826, en die hoogste was 0.25 mg ml-1 na 48 uur. Die minimum inhiberende konsentrasie waardes het gestyg na 0.5mg ml-1 na 60 uur, wat ‘n fungistatiese aksie getoon het. Maar van 60 tot 92 uu het waardes egter ‘n swamdodende aksie aangetoon met geen verandering van 0.5mg ml-1 nie. In die identifisering van die bioaktiewe komponente, is die ekstraksie van Stellenbosch se bevolking in dichloromethan: methanol (1:1 v/v) uitgevoer, en met gebruik van vyftig gram van die ekstrak is bioanalise-geleide fraksionering gedoen deur gebruik van ‘n VersaFlash®. Die mees aktiewe fraksie is verder gefraksioneer deur die gebruik van ‘n konvensionele silikajel kolom. Aktiewe fraksies is getoets deur LC-MS te gebruik, en twee verbindings, carnosol en carnosic suur, is geïdentifiseer. Voorbereidende TLC is gebruik om identiteit te bevestig, want fraksies was naas die kommersiele standaarde van die twee verbindings getoets. Sinergistiese aktiwiteit van die twee samestellings is ondersoek deur ‘n antiswam ontleding teen die vier swamlyne uit te voer. Hierdie studie het dus die veronderstelde bestaan van verskillende chemotipes tussen die bevolkings waarvan voorbeelde geneem is, bekend gestel. Veral die Stellenbosch se bevolking het die meeste verskil, heel moontlik omrede die verlengde en hoër versteurings deur die nabyheid van mense. Plant– omgewing interaksies speel ‘n belangrike rol in die metaboloom van plante, wat dan indirek hul eienskappe verander, en in hierdie geval die antiswam aktiwiteit. Die tuingroeiende bevolking was die mees aktief, heel moontlik omrede hierdie aspek. Nietemin was geen bioaktiwiteit verloor waar die mees kragtige bevolking met weefsel kultuur kondisies in aanraking gebring is nie. Dus is dit ideaal vir kommersialisering. Een nuwe belangrike bevinding was die carnosol en carnosic suur wat twee welbekende samestellings is wat meesal geassosieer is met Rosmarinus officinalis en gedokumenteer is vir antioksidant aktiwiteit. Hier dui laasgenoemde samestellings ‘n antiswam aktiwiteit aan teen die getoetste Fusarium swamlyne. Met ‘n gevestigde weefsel kultuur sisteem alreeds in plek, voorsien dit ‘n beginpunt vir die bestudering van hoe hierdie bioaktiewe komponente in massa geproduseer kan word in die ontwikkeling van nuwe swamdodende produkte.

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