Longitudinal evaluation of dehydroepiandrosterone (DHEA), its sulfated form and estradiol with cancer-related cognitive impairment in early-stage breast cancer patients receiving chemotherapy.

The purpose of this study is to elucidate how patient-reported cognitive symptoms manifest from variations in hormone levels or precursors such as dehydroepiandrosterone (DHEA) and its sulfated form [collectively termed as DHEA(S)] and to investigate their association in breast cancer survivors. Levels of estradiol and DHEA(S) were compared between early-stage breast cancer patients with and without cancer-related cognitive impairment (CRCI) during adjuvant chemotherapy. Data were analyzed from 242 patients (mean age ± SD = 50.8 ± 9.2 years) who had completed FACT-Cog v.3.0, blood draws and questionnaires. Regression model was used to fit the magnitude of change in each respective biomarker levels against overall cognitive impairment status while adjusting for clinically important covariates. There was reduction in mean plasma levels of estradiol and DHEAS during and towards the end of chemotherapy (p-values < 0.001). Compared to non-impaired patients, smaller magnitude of decline was observed in DHEA(S) levels in patients reporting CRCI, with significant association between decline in DHEAS levels and acute onset of CRCI at 6 weeks from baseline (adjusted ß of 0.40, p-value of 0.02). In contrast, patients reporting CRCI showed greater magnitude of decline in estradiol compared to non-impaired patients, although this was not found to be statistically significant. There was an association between magnitude of change in biomarker levels with self-reported CRCI which suggests that the hormonal pathway related to DHEAS may be implicated in acute CRCI for breast cancer survivors. Our findings help to improve biological understanding of the pathway from which DHEAS may correlate with cognitive dysfunction and its impact on cancer survivors.

Differential activation of pregnane X receptor by carnosic acid, carnosol, ursolic acid, and rosmarinic acid.

Pregnane X receptor (PXR) regulates the expression of many genes, including those involved in drug metabolism and transport, and has been linked to various diseases, including inflammatory bowel disease. In the present study, we determined whether carnosic acid and other chemicals in rosemary extract (carnosol, ursolic acid, and rosmarinic acid) are PXR activators. As assessed in dual-luciferase reporter gene assays, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, activated human PXR (hPXR) and mouse PXR (mPXR), whereas carnosol and ursolic acid, but not carnosic acid or rosmarinic acid, activated rat PXR (rPXR). Dose-response experiments indicated that carnosic acid, carnosol, and ursolic acid activated hPXR with EC50 values of 0.79, 2.22, and 10.77µM, respectively. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, transactivated the ligand-binding domain of hPXR and recruited steroid receptor coactivator-1 (SRC-1), SRC-2, and SRC-3 to the ligand-binding domain of hPXR. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, increased hPXR target gene expression, as shown by an increase in CYP3A4, UGT1A3, and ABCB1 mRNA expression in LS180 human colon adenocarcinoma cells. Rosmarinic acid did not attenuate the extent of hPXR activation by rifampicin, suggesting it is not an antagonist of hPXR. Overall, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, are hPXR agonists, and carnosic acid shows species-dependent activation of hPXR and mPXR, but not rPXR. The findings provide new mechanistic insight on the effects of carnosic acid, carnosol, and ursolic acid on PXR-mediated biological effects.

Evaluation of Ginkgo biloba extract as an activator of human glucocorticoid receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba, which is one of the most frequently used herbal medicines, is commonly used in the management of several conditions, including memory impairment. Previously, it was reported to decrease the expression of peripheral benzodiazepine receptor and the biosynthesis of glucocorticoids, thereby regulating glucocorticoid levels. However, it is not known whether Ginkgo biloba extract regulates the function of the glucocorticoid receptor. AIM OF THE STUDY: We determined whether Ginkgo biloba extract and several of its chemical constituents affect the activity of human glucocorticoid receptor (hGR). MATERIALS AND METHODS: A hGR-dependent reporter gene assay was conducted in HepG2 human hepatocellular carcinoma cells and hGR target gene expression assays were performed in primary cultures of human hepatocytes. RESULTS: Multiple lots and concentrations of the extract and several of its chemical constituents (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide) did not increase hGR activity, as assessed by a cell-based luciferase reporter gene assay. The extract did not influence the expression of hGR target genes, including tyrosine aminotransferase (hTAT), constitutive androstane receptor (hCAR), or pregnane X receptor (hPXR), in primary cultures of human hepatocytes. Moreover, hGR antagonism by mifepristone (also known as RU486) did not attenuate the extent of induction of hCAR- and hPXR-regulated target genes CYP2B6 and CYP3A4 by Ginkgo biloba extract. CONCLUSION: Ginkgo biloba extract, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide are not activators of hGR. Furthermore, the extract does not influence the hGR-hCAR or the hGR-hPXR signaling pathway in primary cultures of human hepatocytes.

Isoform-selective activation of human constitutive androstane receptor by Ginkgo biloba extract: functional analysis of the SV23, SV24, and SV25 splice variants.

Naturally occurring splice variants of human constitutive androstane receptor (hCAR) exist, including hCAR-SV23 (insertion of amino acids SPTV), hCAR-SV24 (APYLT), and hCAR-SV25 (SPTV and APYLT). An extract of Ginkgo biloba was reported to activate hCAR-SV24 and the wild type (hCAR-WT). However, it is not known whether it selectively affects hCAR splice variants, how it activates hCAR isoforms, and which chemical is responsible for the effects of the extract. Therefore, we evaluated the impact of G. biloba extract on the functionality of hCAR-SV23, hCAR-SV24, hCAR-SV25, and hCAR-WT and compared it with that of phenobarbital, di-(2-ethylhexyl)phthalate (DEHP), 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in cell-based reporter gene assays. Among the hCAR splice variants investigated, only hCAR-SV23 was activated by G. biloba extract, and this required cotransfection of a retinoid X receptor α (RXRα) expression plasmid. The extract activated hCAR-SV23 to a lesser extent than hCAR-WT, but ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide were not responsible for the effects of the extract. CITCO activated hCAR-SV23, hCAR-SV24, and hCAR-WT. By comparison, phenobarbital activated hCAR-WT, whereas DEHP activated hCAR-SV23, hCAR-SV24 (with exogenous RXRα supplementation), and hCAR-WT. TCPOBOP did not affect the activity of any of the isoforms. G. biloba extract and phenobarbital did not bind or recruit coactivators to the ligand-binding domains of hCAR-WT and hCAR-SV23, whereas positive results were obtained with the controls (CITCO for hCAR-WT and DEHP for hCAR-SV23). In conclusion, G. biloba extract activates hCAR in an isoform-selective manner, and hCAR-SV23, hCAR-SV24, and hCAR-WT have overlapping, but distinct, sets of ligands.

Beta-amyrin from Ardisia elliptica Thunb. is more potent than aspirin in inhibiting collagen-induced platelet aggregation.

Ardisia elliptica Thunberg (Myrsinaceae) is a medicinal plant traditionally used for alleviating chest pains, treatment of fever, diarrhoea, liver poisoning and parturition complications. The objectives of the study were to investigate the effect of A. elliptica on collagen induced platelet aggregation and to isolate and purify potential antiplatelet components. Fresh A. elliptica leaves were extracted using methanol (70% v/v) by Soxhlet extraction and the extract was analysed for its inhibition of collagen-induced platelet aggregation. Inhibition of platelet aggregation was assessed by incubating the extracts with rabbit blood and collagen in a whole blood aggregometer and measuring the impedance. The leaf extract was found to inhibit platelet aggregation with an IC50 value of 167 microg/ml. Using bioassay guided fractionation, beta-amyrin was isolated and purified. The IC50 value of beta-amyrin was found to be 4.5 microg/ml (10.5 microM) while that of aspirin was found to be 11 microg/ml (62.7 microM), indicating that beta-amyrin was six times as active as aspirin in inhibiting platelet aggregation. This paper is the first report that beta-amyrin isolated from A. elliptica is more potent than aspirin in inhibiting collagen-induced platelet aggregation. In conclusion, A. elliptica leaves were found to inhibit collagen-induced platelet aggregation and one of the bioactive components responsible for the observed effect was determined to be beta-amyrin.

Human pregnane X receptor agonism by Ginkgo biloba extract: assessment of the role of individual ginkgolides.

Ginkgo biloba extract activates pregnane X receptor (PXR), but how this occurs is not known. Therefore, we investigated the mechanism of PXR activation by the extract and the role of five individual terpene trilactones in the activation. In a cell-based reporter gene assay, G. biloba extract activated human PXR (hPXR), and at a concentration present in the extract, ginkgolide A, but not ginkgolide B, ginkgolide C, ginkgolide J, or bilobalide was partially responsible for the increase in hPXR activity of the extract. Likewise, in cultured human hepatocytes, only ginkgolide A contributed to the increase in hPXR target gene expression (CYP3A4 mRNA and CYP3A-mediated testosterone 6ß-hydroxylation). The extract, but none of the terpene trilactones, bound to hPXR ligand-binding domain, as analyzed by a time-resolved fluorescence resonance energy transfer competitive binding assay. Only the extract and ginkgolide A recruited steroid receptor coactivator-1, as determined by a mammalian two-hybrid assay. Compared with hPXR, rat PXR (rPXR) was activated to a lesser extent by G. biloba extract. Similar to hPXR, only ginkgolide A contributed to rPXR activation by the extract. In contrast to the effect of G. biloba extract on PXR function, it did not affect hPXR expression. Overall, the main conclusions are that G. biloba extract is an hPXR agonist, and among the five terpene trilactones investigated, only ginkgolide A contributes to the actions of the extract. Our findings provide insights into the biological and chemical mechanisms of hPXR activation by G. biloba extract.

Antiplatelet and anticoagulant effects of Panax notoginseng: comparison of raw and steamed Panax notoginseng with Panax ginseng and Panax quinquefolium.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Panax notoginseng (Burk.) F. H. Chen (Araliacea) is traditionally used for its hemostatic and cardiovascular effects when raw and as a tonic when steamed. AIM OF THE STUDY: This study aims to compare the effects of raw and steamed Panax notoginseng, Panax ginseng C. A. Meyer and Panax quinquefolium Linn. on platelet aggregation and plasma coagulation. MATERIALS AND METHODS: Effects on collagen-induced platelet aggregation were investigated using a platelet aggregometer, while the plasma coagulation times (prothrombin time, activated partial thromboplastin time and thrombin time) were determined using a blood coagulation analyzer. The data was corroborated with ex vivo platelet aggregation and in vivo rat bleeding time. RESULTS: Raw and steamed Panax notoginseng significantly inhibit platelet aggregation and plasma coagulation. Steamed Panax notoginseng has significantly more potent antiplatelet and anticoagulant effects than the raw extract, and the antiplatelet and anticoagulant effects increase with increasing steaming durations. Comparing the three common Panax species, Panax notoginseng has higher antiplatelet effect than Panax ginseng and Panax quinquefolium. The in vitro antiplatelet and anticoagulant effects are positively translated into a prolongation of in vivo rat bleeding time after oral administration of the raw and steamed extracts. CONCLUSION: The results indicate that the three common Panax species affect platelet aggregation and plasma coagulation differently, with steamed Panax notoginseng showing the greatest antiplatelet and anticoagulant effects. Panax notoginseng may be a good source of lead compounds for novel antiplatelet and anticoagulant therapeutics.

Inhibition of human CYP2B6-catalyzed bupropion hydroxylation by Ginkgo biloba extract: effect of terpene trilactones and flavonols.

Cytochrome P450 2B6 (CYP2B6) is expressed predominantly in human liver. It catalyzes the oxidative biotransformation of various drugs, including bupropion, which is an antidepressant and a tobacco use cessation agent. Serious adverse effects of high dosages of bupropion have been reported, including the onset of seizure. As Ginkgo biloba extract may be consumed with bupropion or another CYP2B6 drug substrate, potential exists for an herb-drug interaction. Therefore, we investigated the effect of G. biloba extract and some of its chemical constituents (terpene trilactones and flavonols) on the in vitro catalytic activity of CYP2B6 as assessed by the bupropion hydroxylation assay with recombinant enzyme and hepatic microsomes. The amount of hydroxybupropion was quantified by a novel and validated ultraperformance liquid chromatography/mass spectrometry method. Enzyme kinetic analysis indicated that G. biloba extract competitively inhibited hepatic microsomal CYP2B6-catalyzed bupropion hydroxylation (apparent K(i) was 162 +/- 14 microg/ml). Bilobalide and ginkgolides A, B, C, and J were not responsible for the inhibition of CYP2B6 catalytic activity by the extract. Whereas the 3-O-glucoside and 3-O-rutinoside of quercetin, kaempferol, and isorhamnetin had no effect, the corresponding aglycones (10 and 50 microg/ml) decreased hepatic microsomal bupropion hydroxylation. The inhibition of CYP2B6 by kaempferol was competitive (apparent K(i) was 10 +/- 1 microg/ml). In summary, G. biloba extract and its flavonol aglycones are naturally occurring inhibitors of in vitro CYP2B6 catalytic activity and bupropion hydroxylation. Future studies are needed to investigate whether G. biloba extract interacts in vivo with bupropion or other clinically important CYP2B6 drug substrates.

Ultra-performance liquid chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed Panax notoginseng.

At present, metabolite profiling is of growing importance in herbal medicine fields such as breeding, formulation, quality control and clinical trials. This preliminary study indicated that ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS)-based metabolomics allows direct detection of down-stream derivatives of metabolites, arising from the herbal formulation process. This analytical approach allows the discrimination and tentative authentication of unique biomarkers related to different herbal extracts using unsupervised multivariate principal component analysis (PCA). The tentative identification of biomarkers is complemented significantly by the accurate mass measurement of TOFMS and the high resolution and high retention time reproducibility rendered by UPLC. The application of this approach in herbal extract discrimination and ginsenoside biomarker discovery of raw and steamed Panax notoginseng (Burk.) F.H. Chen is demonstrated and discussed.

Hydrophilic interaction liquid chromatography with tandem mass spectrometry for the determination of underivatized dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) in Panax medicinal plant species.

Dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) is a haemostatic agent present in important Chinese medicinal herbs such as Panax notoginseng, as well as other Panax species. It is also a reported neurotoxic agent found in Lathyrus sativus (grass pea seed). A selective analytical method incorporating hydrophilic interaction chromatography with positive electrospray ionization tandem mass spectrometry (HILIC/ESI-MS/MS), for the analysis of dencichine in Panax plant species, was developed. Using multiple reaction monitoring (MRM) mode, underivatized dencichine, a small and highly polar compound, was selectively detected and quantified. The contents of dencichine in raw and steamed Panax notoginseng roots, 11 pairs of raw and steamed P. notoginseng herbal products, Panax ginseng roots, and Panax quinquefolium roots, were analyzed and compared. Optimal sensitivity of 0.3 ppm (detection limit) and 1.5 ppm (quantification limit) was achieved. The method was rapid (< or =5 min), with the HILIC peak eluting at about 1 min. Steamed P. notoginseng samples were found to contain less dencichine than the corresponding raw samples, and there were also differences among the three Panax species; raw P. ginseng and P. quinquefolium contained less dencichine than the raw P. notoginseng species. This rapid and specific method may be applied to the quantification of dencichine in complex medicinal plants and their products.

High-performance liquid chromatographic method with quantitative comparisons of whole chromatograms of raw and steamed Panax notoginseng.

A high-performance liquid chromatographic (HPLC) method coupled with chromatographic pattern matching was developed to differentiate whole chromatograms of raw and steamed Panax notoginseng objectively and quantitatively. The major peaks differentiating chromatograms of raw and steamed samples were also identified for the first time in this herb. The raw and steamed P. notoginseng roots and its products were successfully differentiated. The quantitative differences between the chromatograms were correlated to the duration of steaming. Chromatographic pattern matching allows rapid, simple, automated, and quantitative comparisons of complex chromatograms. It is a useful tool in ensuring safety and quality of herbal products.

Analysis of saponins in raw and steamed Panax notoginseng using high-performance liquid chromatography with diode array detection.

A reversed-phase high-performance liquid chromatography-diode array detection method was developed and validated for the simultaneous determination of six saponins (notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rc, Rd) in raw and steamed Panax notoginseng. Linearity (r2 > 0.9988), intra- and inter-day precision (RSD < 4%), limit of detection (0.008-0.013 mg/ml), limit of quantification (0.027-0.042 mg/ml) of the saponins were determined. The method was successfully applied to 11 pairs of raw and steamed P. notoginseng products. Three products showed discrepancies between theirlabelled claims (raw or steamed) and the results of analysis. This new, simple and reliable method could be used in the quality control of raw and steamed P. notoginseng.

Analysis of adulterants in a traditional herbal medicinal product using liquid chromatography-mass spectrometry-mass spectrometry.

Adulterations with synthetic drugs are common problems with herbal medicine and this can potentially cause serious adverse effects. It is therefore important to determine the presence of synthetic drugs in herbal medicine to ensure patients' safety. The objective of this study was to develop sensitive and specific methods to analyse phenylbutazone, caffeine and oxyphenbutazone present in a traditional Indonesian herbal product. Liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) methods in the selected reaction-monitoring (SRM) mode were developed. It was found that the sample contained 0.53% w/w (n=3, RSD=7.56%) phenylbutazone and 0.04% w/w (n=3, RSD=8.39%) caffeine. This corresponded to 43.17 mg phenylbutazone and 3.23 mg caffeine in each sachet of powder. The methods were validated for linearity, precision, accuracy, LOD and LOQ. LOD and LOQ were found to be 3.69 and 12.29 ng/ml, respectively for phenylbutazone. For caffeine, the LOD and LOQ were 0.84 and 2.80 ng/ml, respectively. Oxyphenbutazone in the sample was found to be present at a level below the quantification level of 10.2 ng/ml. With better methods developed for analysis of adulterants in herbal medicine, the quality and safety of these medicines can be better controlled and regulated to ensure patients' safety.