Ann Louw

South African herbal teas: Aspalathus linearis, Cyclopia spp. and Athrixia phylicoides—A review

Rooibos (Aspalathus linearis (Brum.f) Dahlg.) and honeybush (Cyclopia Vent. species) are popular indigenous South African herbal teas enjoyed for their taste and aroma. Traditional medicinal uses of rooibos in South Africa include alleviation of infantile colic, allergies, asthma and dermatological problems, while a decoction of honeybush was used as a restorative and as an expectorant in chronic catarrh and pulmonary tuberculosis. Traditional medicinal uses of Athrixia phylicoides DC., or bush tea, another indigenous South African plant with very limited localised use as herbal tea, include treatment of boils, acne, infected wounds and infected throats. Currently rooibos and honeybush are produced for the herbal tea market, while bush tea has potential for commercialisation. A summary of the historical and modern uses, botany, distribution, industry and chemical composition of these herbal teas is presented. A comprehensive discussion of in vitro, ex vivo and in vivo biological properties, required to expand their applications as nutraceutical and cosmeceutical products, is included, with the main emphasis on rooibos. Future research needs include more comprehensive chemical characterisation of extracts, identification of marker compounds for extract standardisation and quality control, bioavailability and identification of bio-markers of dietary exposure, investigation of possible herb-drug interactions and plant improvement with regards to composition and bioactivity.

Ligand-selective transactivation and transrepression via the glucocorticoid receptor: Role of cofactor interaction

The mechanisms that determine ligand-selective transcriptional responses by the glucocorticoid receptor (GR) are not fully understood. Using a wide panel of GR ligands, we investigated the relationships between the potency and maximal response for transactivation via a glucocorticoid response element (GRE) and transrepression via both nuclear factor small ka, CyrillicB (NFsmall ka, CyrillicB) and activator protein-1 (AP-1) sites, relative binding affinity for the GR, as well as interaction with both coactivators and corepressors. The results showed ligand-selective differences in potency and efficacy for each promoter, as well as for a particular ligand between the three promoters. Ligand potency correlated with relative affinity for the GR for agonists and partial agonists in transactivation but not for transrepression. Maximal response was unrelated to relative affinity of ligand for GR for both transactivation and transrepression. A good and significant correlation between full length coactivator binding in two-hybrid assays and efficacy as well as potency of different receptor-steroid complexes for both transactivation and transrepression supports a major role for coactivator recruitment in determination of ligand-selective transcriptional activity. Furthermore, ligand-selective GR binding to GRIP-1, as determined by both two-hybrid and DNA pull down assays, correlated positively with ligand-selective efficacy for transactivation of both a synthetic GRE reporter with expressed GR as well as of an endogenous gene via endogenous GR. The receptor interacting domain of the corepressor SMRT exhibited strong interaction with both agonists and partial agonists, similar to the results for coactivators, suggesting a possible role for SMRT in activation of transcription. However, there was no correlation between ligand affinity for the GR and cofactor interaction. These results provide strong quantitative biochemical support for a model in which GR-mediated ligand-selective differential interaction with GRIP-1, SRC-1A, NCoR and SMRT is a major determinant of ligand-selective and promoter-specific differences in potency and efficacy, for both transactivation and transrepression.

Abrogation of Glucocorticoid Receptor Dimerization Correlates with Dissociated Glucocorticoid Behavior of Compound A

Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [3H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GRdim) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.

Mechanism for the stabilization in vivo of the aziridine precursor 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride by serum proteins

Oral and intraperitoneal administration of 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound A), an analogue of phenyl aziridine precursors that occur in the shrub Salsola tuberculatiformis Botsch, had a contraceptive effect on female Wistar rats with a concomitant decrease in total body, uterus, and every mass and an increase in abronal mass. Compound A elicited a Type II difference spectrum and inhibited the Type I deoxycorticosterone (DOC) induced difference spectrum of sheep adrenal cytochrome P450c11 in a manner similar to that of S2, a biologically active fraction isolated from S. tuberculatiformis. The effects of Compound A on the spectral properties of P450c11 were diminished with time in PBS. Electrospray mass spectrometry (ES-MS) indicated that the rate of cyclization of Compound A to the corresponding aziridine followed a time course similar to the attenuation of cytochrome P450c11 inhibition. It was concluded that the aziridine precursor. Compound A, rather than aziridine itself, was the inhibiting agent of sheep adrenal P450c11. Addition of sheep and rat plasma prevented the attenuation of the effect of Compound A on the spectral properties of cytochrome P450c11. Subsequent ES-MS analysis indicated that Compound A was stabilized in plasma by sex hormone binding globulin and corticosteroid binding globulin. These results suggest a mechanism whereby natural plant products, which are highly reactive and unstable in vitro, can be stabilized by binding to plasma proteins, and so remain biologically active in vivo.

Role of ligand-dependent GR phosphorylation and half-life in determination of ligand-specific transcriptional activity☆

A central question in glucocorticoid mechanism of action via the glucocorticoid receptor (GR) is what determines ligand-selective transcriptional responses. Using a panel of 12 GR ligands, we show that the extent of GR phosphorylation at S226 and S211, GR half-life and transcriptional response, occur in a ligand-selective manner. While GR phosphorylation at S226 was shown to inhibit maximal transcription efficacy, phosphorylation at S211 is required for maximal transactivation, but not for transrepression efficacy. Both ligand-selective GR phosphorylation and half-life correlated with efficacy for transactivation and transrepression. For both expressed and endogenous GR, in two different cell lines, agonists resulted in the greatest extent of phosphorylation and the greatest extent of GR downregulation, suggesting a link between these functions. However, using phosphorylation-deficient GR mutants we established that phosphorylation of the GR at S226 or S211 does not determine the rank order of ligand-selective GR transactivation. These results are consistent with a model whereby ligand-selective GR phosphorylation and half-life are a consequence of upstream events, such as ligand-specific GR conformations, which are maintained in the phosphorylation mutants.

Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model.

Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.

Influence of an Aziridine Precursor on the In Vitro Binding Parameters of Rat and Ovine Corticosteroid- Binding Globulin (CBG)

Aziridines are highly reactive alkylating compounds used in cancer treatment. Salsola tuberculatiformis Botsch., which causes prolonged gestation in sheep and contraception in rats, contains a very labile hydroxy-phenylaziridine or its precursor. A less labile analogue, 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound I), was synthesized and has been shown to be contraceptive in rats and to be stabilized by corticosteroid-binding globulin (CBG). The current study compared the binding parameters of rat and ovine CBG and evaluated the effect of the aziridine precursor, Compound I, on these parameters. Kd and Bmax values of 0.646 and 578 nM for corticosterone binding to rat CBG and 0.577 and 19.8 nM for cortisol binding to sheep CBG, respectively, were measured. In competitive binding studies with rat plasma, Ki values of 3.48 nM, 0.856 nM, 22.2 nM, 722 microM, and > 1,000,000 microM for cortisol, corticosterone, progesterone, Compound I, and synephrine (Compound II), respectively, were found, while in sheep plasma the values were 0.409 nM, 1.78 nM, 5.28 nM, 594 microM, and > 1,000,000 microM, respectively. Concentrations of Compound I equivalent to an effective pharmacological dose resulted in a significant (P < 0.01) decrease in CBG bound corticosterone and a significant (P < 0.01) increase in free corticosterone in rat plasma. In sheep, a similar effect was observed with cortisol. Progesterone binding, however, did not appear to be affected significantly by Compound I in either rat or sheep plasma. Compound I was found to be a competitive inhibitor of glucocorticoid binding to CBG. These results suggest that binding of Compound I to CBG with concomitant displacement of endogenous glucocorticoids, but not progesterone, may be part of the mechanism of action of these phenylaziridine compounds.

Anti-androgenic properties of Compound A, an analog of a non-steroidal plant compound

We investigated the interactions between Compound A (CpdA), an analog of a hydroxyphenyl aziridine precursor found in an African shrub, and the androgen receptor (AR). CpdA represses androgen-induced activation of both specific and non-specific androgen DNA response elements. While a similar effect was obtained for the progesterone receptor (PR) via a non-specific hormone response element, CpdA had no effect on the actions of the glucocorticoid and mineralocorticoid receptors. CpdA represses the ligand-dependent interaction between the NH(2)- and COOH-terminal domains of the AR, similar to well-characterised anti-androgens. CpdA also interferes with the interaction of steroid receptor co-activator 1 (SRC1) with the activation domain AF2 but not with AF1. However, CpdA does not compete with androgen for binding to the AR. These results demonstrate that CpdA elicits anti-androgenic actions by a mechanism other than competitive binding for the AR.

Phytoestrogenic potential of Cyclopia extracts and polyphenols

Cyclopia Vent. species, commonly known as honeybush, are endemic to Southern Africa. The plant is traditionally used as an herbal tea but several health benefits have recently been recorded. This minireview presents an overview of polyphenols found in Cyclopia and focuses on the phytoestrogenic potential of selected polyphenols and of extracts prepared from the plant.

Interplay of the Inflammatory and Stress Systems in a Hepatic Cell Line: Interactions between Glucocorticoid Receptor Agonists and Interleukin-6

The liver plays an important role in inflammation and stress by producing the acute phase proteins (APPs) required for resolution of inflammation as well as by delivering systemic glucose, through gluconeogenesis, required to fuel the stress response. Disruption of the interplay between interleukin 6 (IL-6) and glucocorticoids (GCs), the peripheral mediators of inflammation and stress, respectively, may lead to side-effects associated with the pharmacological use of GCs. The current study investigated the interplay between IL-6 and GCs in a hepatoma cell line (BWTG3) at protein (protein activity assays, Western blotting, and ELISA) and mRNA (qPCR) levels. Specifically, the action of dexamethasone (Dex), a known antiinflammatory drug and glucocorticoid receptor (GR) agonist, is compared to that of Compound A (CpdA), a selective glucocorticoid receptor agonist (SEGRA). CpdA, like IL-6, but unlike Dex, increases GR binding and decreases the metabolic enzymes, tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and gamma glutamyltransferase, at protein or mRNA level. Like Dex, both CpdA and IL-6 increase the positive APPs, serum amyloid A and C-reactive protein, and decrease the negative APP, corticosteroid binding globulin. The study shows that the GC, Dex, and IL-6 generally have divergent effects on the GR and metabolic enzymes, while their functions are convergent on the APPs. In contrast to Dex, CpdA has effects convergent to that of IL-6 on the GR, metabolic enzymes, and APPs. Thus these findings suggest that CpdA, like Dex, modulates APPs, leading to effective control of inflammation, while, in contrast to Dex, it is less likely to lead to GC-induced side-effects.