Investigating the parts to understand the whole : contribution of phenolic compounds to the desired estrogenic activity of SM6Met fractions from Cyclopia subternata

Claassen, Herzelle (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Decreasing estrogen levels, associated with menopause, may cause several unpleasant symptoms. Hormone replacement therapy (HRT) relieves these symptoms, but increases the risk of developing breast cancer. As estrogens promote estrogen-receptor positive (ER+) breast cancer, women are seeking alternative forms of hormone therapy and are turning to plant-based products high in phytoestrogens, since they are considered to be a more natural, healthier and potentially safer alternative to HRT. Another alluring aspect of plant-based products are their ability to exert their effect through multiple target mechanisms, which is potentially due to the complex mixture of different bio-active and supporting compounds working together. The current study forms part of a larger ongoing study, which focuses on Cyclopia, an indigenous South African fynbos plant, as a potential nutraceutical to reduce menopausal symptoms, while decreasing the risk of breast cancer. The sequential methanol extract of Cyclopia subternata (SM6Met) displayed promising results in different in vitro and in vivo breast cancer experimental systems, with the desired estrogenic characteristics of ER subtype selectivity, specifically ERβ agonism and ERα antagonism (which is important in ER+ breast cancer since ERα is known to induce proliferation, whereas ERβ antagonises ERα), and the inhibition of estrogen-induced breast cancer cell proliferation. However, the compounds responsible for the desired estrogenic characteristics have not been identified and thus, as a starting point, we focused on the 7 major phenolic compounds quantified in a fraction, F3, obtained from SM6Met. F3 previously displayed robust ERβ agonism, and the aim of the current study was thus to evaluate the combinatorial effect of these 7 major phenolic compounds to the favourable estrogenic profile of F3. Firstly, an experimental system sensitive enough to detect small drug induced changes was successfully established, after which the phytoestrogenic activity of the 7 major phenolic compounds were tested individually and in combination. Interestingly, the reconstituted faction (F3R), in contrast to F3, displayed no significant estrogenic activity when the 7 major phenolic compounds were reconstituted at a concentration equivalent to that which is present in F3. However, when F3R was reconstituted at 100-fold its concentration, a significant increase in activity was observed via both ER subtypes. Mangiferin and luteolin were the only two phenolic compounds that displayed significant and dose-dependent l estrogenic activity when tested alone. Since, at this stage of the study, not much was known regarding the estrogenicity of mangiferin, a more in detailed investigation revealed activity via ERα and also, as a novel finding, via ERβ. The current study concluded that F3’s robust ERβ agonist activity could not be recreated using only the 7 major phenolic compounds at their concentration in F3 but that the minor phenolic compounds play a crucial supporting role in the desired estrogenic profile of this fraction. Thus, the contribution of the 7 major phenolic compounds to F3’s activity, and subsequently the multi target activity of SM6Met, should not be dismissed, since other compounds present in F3, but not in F3R, may act synergistically with the major phenolic compounds to produce the robust ERβ activity of F3.

AFRIKAANSE OPSOMMING: Die vermindering van estrogeenvlakke, wat met menopouse gepaard gaan, kan verskeie onaangename simptome veroorsaak. Hormoon-vervangingsterapie (HVT) verlig hierdie simptome, maar verhoog die risiko om borskanker te ontwikkel. Aangesien estrogeen-reseptor positiewe (ER+) borskanker deur estrogeen bevorder word, soek vrouens alternatiewe vorme van hormoonterapie en wend hulle na plantgebaseerde produkte wat hoog is in fitoestrogene, aangesien fitoestrogene beskou word as 'n meer natuurlike, gesonder en potensieel veiliger alternatief teenoor HVT. Nog 'n aanloklike aspek van plantgebaseerde produkte is hul vermoë om hul effek uit te oefen deur verskeie teiken meganismes, wat moontlik te wyte is aan die komplekse mengsel van verskillende bio-aktiewe en ondersteunende verbindings wat saamwerk. Die huidige studie vorm deel van 'n groter deurlopende studie wat fokus op Cyclopia, 'n inheemse Suid-Afrikaanse fynbosplant, as 'n potensiële nutraceutiese middel om menopousale simptome te verminder, terwyl die risiko van borskanker verlaag word. Die sekwensiële metanol ekstrak van Cyclopia subternata (SM6Met) het belowende resultate in verskeie in vitro en in vivo borskanker eksperimentele stelsels getoon, met die gewensde estrogeen eienskappe van ER subtypeselektiwiteit, spesifiek ERβ-agonisme en ERα-antagonisme (wat belangrik is in ER+ borskanker, aangesien ERα bekend is om proliferasie te bewerkstellig, terwyl ERβ vir ERα antagoniseer) en die inhibisie van estrogeen-geïnduseerde borskanker proliferasie. Die verbindings wat verantwoordelik is vir die gewensde estrogeen eienskappe is egter nog nie geïdentifiseer nie, en dus as ‘n begin punt, word daar gefokus op die 7 hoof fenoliese verbindings wat gekwantifiseer is in 'n fraksie, F3, verkry uit SM6Met. F3 het voorheen robuuste ERβ agonisme getoon, en die doel van die huidige studie was dus om die kombinatoriese effek van hierdie 7 hoof fenoliese verbindings te evalueer op die gunstige estrogeenprofiel van F3. Eerstens was 'n eksperimentele stelsel daargestel wat sensitief genoeg is om klein middel-geïnduceerde veranderings suksesvol op te tel, waarna die fitoestrogeen aktiwiteit van die 7 hoof fenoliese verbindings individueel en in kombinasie getoets is. Interessant genoeg, het die hersaamgestelde fraksie (F3R), in teenstelling met F3, geen beduidende estrogeen aktiwiteit getoon nie toe die 7 hoof fenoliese verbindings gekombineer is by 'n konsentrasie gelykstaande aan wat in F3 teenwoordig is. Toe F3R egter teen 100-voudige konsentrasie hersaamgestel is, is 'n beduidende toename in aktiwiteit waargeneem via beide ER subtipes. Mangiferin en luteolin was die enigste twee fenoliese verbindings wat beduidende en dosis afhanklike estrogeen aktiwiteit getoon het toe hulle alleen getoets is. Aangesien daar op hierdie stadium van die studie nie veel bekend was omtrent die estrogenisiteit van mangiferin nie, het 'n meer gedetailleerde ondersoek aktiwiteit via ERα, en ook as 'n nuwe bevinding via ERβ, aan die lig gebring. Die huidige studie het tot die gevolgtrekking gekom dat F3 se robuuste ERβ-agonistiese aktiwiteit nie herskep kon word deur slegs die 7 hoof fenoliese verbindings by hul konsentrasie in F3 te gebruik nie, maar dat die ander fenoliese verbindings 'n belangrike ondersteunings rol speel in die gewenste estrogeenprofiel van hierdie fraksie. Dus, moet die bydrae van die 7 hoof fenoliese verbindings tot F3 se aktiwiteit, en die daaropvolgende multi-doelaktiwiteit van SM6Met, nie gering geskat word nie, aangesien ander verbindings teenwoordig in F3, maar nie in F3R, sinergisties mag optree met die hoof fenoliese verbindings om die robuuste ERβ aktiwiteit van F3 te produseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103890
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