Dietetics, Nutrition and Biological Sciences
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Item The Inhibition of Free Radical Generation by Preparations of Harpagophytum procumbens in vitro(2009-01) Grant, L.; McBean, Douglas; Fyfe, Lorna; Warnock, MaryHarpagophytum procumbens (Hp), commonly known as Devil's Claw has been used as a traditional treatment for a variety of illnesses for centuries. Since the early twentieth century, it has become a popular antiinflammatory and analgesic preparation amongst European herbalists for supportive or adjuvant treatment of degenerative joint diseases. Extracts of Hp tubers have demonstrated antiinflammatory and analgesic effects in animal models of inflammation and in human trials. The mechanism(s) of action responsible for these attributes, however, remain to be elucidated. Reactive oxygen species generated in acute and chronic inflammatory diseases are known to be cytotoxic and can cause tissue damage. In this study, a root tuber extract (Hp extract) and commercially available tincture (Hp tincture) were investigated for antioxidant characteristics using in vitro test systems. Both preparations were found to effectively scavenge DPPH radical, inhibit nitrite levels in supernatants harvested from LPS-stimulated RAW 264.7 macrophages, and cause dose-dependent suppressions in the detection of fMLP-and AA-induced neutrophil MPO. The antioxidant effects demonstrated for both preparations of Hp may contribute to the antiinflammatory and analgesic properties observed for the plant. Copyright © 2008 John Wiley & Sons, LtdItem Honey: A realistic antimicrobial for disorders of the skin(2015-01-19) McLoone, Pauline; Warnock, Mary; Fyfe, LornaResistance of pathogenic micro-organisms to antibiotics is a serious global health concern. In this review, research investigating the antimicrobial properties of honeys from around the world, against skin relevant microbes is evaluated. A plethora of in vitro studies have revealed that honeys from all over the world have potent microbicidal activity against dermatologically important microbes. Moreover, in vitro studies have shown that honey can reduce microbial pathogenicity as well as reverse anti-microbial resistance. Studies investigating the antimicrobial properties of honey in vivo have been more controversial. It is evident that innovative research is required to exploit the anti-microbial properties of honey for clinical use and to determine the efficacy of honey in the treatment of a range of skin disorders with a microbiological aetiologyItem Physiological levels of soy isoflavones reduce proliferation and promote apoptosis in the ER_-positive breast cancer cell line MDA-MB-231(Cambridge University Press, 2012-04) Wallace, J.; Warnock, Mary; Gow, Iain F.Dietary soy, particularly the high levels consumed as part of traditional Eastern-Asian diets, may be protective against certain subtypes of breast cancer(1). This effect is partly related its high isoflavone phytoestrogen content (genistein and daidzein). However much in vitro evidence suggests a contradictory, growth promoting impact of physiological (serum) levels of isoflavones in oestrogen receptor alpha positive (ERa+) breast cancer cell lines(2). This has led to the contraindication of isoflavone supplement use in post menopausal breast cancer survivors. The inconsistency between epidemiological and in vitro evidence may relate to the relative expression levels of ERa and its counterpart ERb. Unlike in cell lines predominantly expressing ERa, the proliferation of ERb-dominant cell lines can be inhibited by concentrations of genistein as low as 1 nM(3,4). This information is relevant as the ERa - /b+ subgroup represent approximately 18% of all breast tumours(5) but the beta-receptor is not routinely tested for upon diagnosis. We assessed the impact of physiologically relevant concentrations (serum levels achievable through diet: 0.01nM to 31.6 mM) of the soy isoflavones genistein and daidzein on proliferation (MTT assay) and apoptosis with the Annexin V-Cy32 Apoptosis Detection Kit and DAPI staining/Nuclear Area Factor (NAF) calculation in the ERa - /b+ breast cancer cell line MDA-MB-231. Low doses of genistein or daidzein (- 0.01 nM) inhibited MDA-MB-231 proliferation by around 20%, although this frequently failed to achieve statistical significance. The highest concentrations used (31.6 mM) resulted in a dramatic decrease in percent proliferation compared with a vehicle only control (meanSD : 48.512.6, p = 0.004 and 48.413.1, p<0.001 for genistein and daidzein respectively; n = 3). 17b-oestradiol (E2; the major circulating oestrogen) also slightly reduced proliferation at its pre- and post-menopausal serum concentrations (1nM and 1 pM respectively). This reduction in proliferation was associated with an observed increase in cell death at 10 and 31.6 mM genistein and daidzein, which was at least partly explained by an increase in the percentage of apoptotic cells. While the combination of E2 and isoflavones failed to show any synergistic growth inhibitory, or pro-apoptotic effects, some inhibition of proliferation was still documented. This implies that the growth inhibitory effects of genistein, daidzein and E2 may not be regulated by the same mechanisms. However, more importantly, the apoptotic cell death observed with soy isoflavone treatment was not abrogated by the addition of physiological E2 concentrations. Overall, our data suggests that at concentrations achievable through the diet ( 10 mM) soy isoflavones may be potentially beneficial as chemotherapeutic agents against ERa - /b+ breast cancers, through their ability to reduce proliferation and promote apoptosis, even in the presence of physiological E2 levels. Routine determination of the expression levels of ERb in addition to ERa in breast cancer would be an invaluable biomarker to indicate the potential efficacy of this cheap and readily available treatment, and indicates a possible pharmacological target. 1. Wu AH, Yu MC, Tseng CC, Pike MC (2008) Brit J Cancer 98:9-14. 2. Hwang CS, Kwak HS, Lim HJ, et al. (2006) J Steroid Biochem Mol Bio 101:246-53. 3. Rajah TT, Du N, Drews N, Cohn R (2009) Pharmacology 84:68-73. 4. Kang X, Jin S, Zhang Q (2009) J Food Sci 74:H237-H242. 5. Skliris GP, Leygue E, Watson PH, Murphy LC (2008) J Steroid Biochem Mol Bio 109:1-10.Item The effect of 17-β oestradiol, resveratrol, and genistein on Na+/H+ exchange in breast cancer cells lines(Cambridge University Press, 2012-03) Wallace, J.; Foy, Daniel; Yousuf, Huma; Warnock, Mary; Gow, Iain F.The tumour environment is more acidic than normal (pH 6.8 vs 7.3)(1), and this may aid tumour progression or affect the uptake of drugs. The extracellular pH may be partly-regulated by cellular sodium/hydrogen exchangers (NHEs), and NHE activation may in turn be regulated by hormones such as oestradiol(2). Some breast cancers possess receptors for oestradiol, and stimulation or blockade of these receptors may modulate the cell's ability to regulate pH. We studied this by investigating the effect of oestradiol on the ability of breast cancer cell lines to regulate pH by NHE. We used MCF-7 (expresses oestrogen receptors a and b) and MDA-MB-231 (expresses only oestrogen receptor b) cell lines. Given the interaction of phytoestrogens with the oestrogen receptor(3), we also looked at the effect of resveratrol (RSV) or genistein (Gen). MCF-7 and MDA-MB-231 cells were used either as suspensions, or on coverslips, in the absence of added oestradiol. Cells were loaded with H+ -sensitive fluorescent probe BCECF, then washed to remove excess dye. Suspended cells were allowed to adhere to the glass bottom of the 35mm perfusion chamber, coverslips with cells were added to the chamber directly. Cells were perfused (1 ml/min) at 37C with HEPES-buffed Tyrode (pH 7.4) to which drugs were added as required. Acidification was induced by the NH4Cl pre-pulse technique, recovery was achieved by returning to normal Tyrode. Cells were illuminated by alternating wavelengths of 440 and 503 nm light (2 Hz sample rate), and fluorescent light (>535 nm) was captured by a CCD camera. The fluorescence ratios are directly proportional to the H+ concentration. Drugs were added directly to the Tyrode, and since RSV and Gen were dissolved in DMSO, DMSO (0.01% final) was added to the pre-perfusing Tyrode in those experiments, and used as a control in the absence of drugs. Results are mean ratio valuesSEM or slopes of rate of change of ratio/minSEM for three separate experimental days, except for the DMSO controls where n = 6. Differences were assessed by Student's paired or unpaired t tests. A brief (4 min) exposure of breast cancer cells to NH4Cl (20mM) caused a transient alkalisation (decrease in 440/503 ratio, i.e. decrease in [H + ]i) followed by an acidification and recovery after ammonium removal. In MDA cells, addition of 17b-oestradiol (E2; 1 pM final concentration) had no effect on the rate of recovery from acidification (Control: - 3.44E-041.34E-04; MDA: - 2.26E-040.69E-04). In MCF-7 cells, perfusing with Tyrode containing 1 pM E2 significantly increased the rate of recovery from acidification (Control: - 1.61E-040.22E-04; MCF-7: - 3.66E- 040.18E-04; p<0.05). Inclusion of DMSO alone in the medium perfusing MCF-7 cells significantly increased the basal ratio compared with cells in Tyrode alone (Control: 1.240.18; DMSO: 1.710.09; p<0.05), and also increased the rate of recovery (Control: - 1.61E- 040.22E-04; DMSO: - 3.97E-040.52E-04; p<0.02). Inclusion of RSV (1 mM) in the perfusate with MCF-7 cells also caused an increase in the rate of recovery relative to the DMSO-only control (DMSO: - 3.97E-040.52E-04; RSV: - 6.84E-040.56E-04; p<0.02). Gen (1 mM) had no significant effects in MCF-7 cells, though all three recovery rates were numerically higher than the DMSO control. In summary, we have shown that E2 and RSV at physiological levels can increase the rate of recovery from acidification in a breast cancer cell line expressing oestrogen receptors a and b. Given the potential importance of cytosolic and extracellular pH regulation in both the ability of cytotoxic drugs to cross the membrane or the cell to proliferate, the physiological effects of foodborne phytohormones on pathways regulating cell pH clearly requires further investigation. 1. Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Taylor CW, Paine-Murrieta G, Roe D, Bhujwalla ZM, Gillies RJ (1999) Br J Cancer 80: 1005-1011. 2. Kilic A, Jaradov S, and Karmazyn M (2009) J Mol Cell Cardiol 46:360-36. 3. Hwang CS, Kwak HS, Lim HJ, Lee SH, Kang YS, Choe TB, Hur HG & Han KO (2006) J Steroid Biochem Mol Biol 101: 246-253.Item A review of the biological and potential therapeutic actions of harpagophytum procumbens(John Wiley & Sons, Ltd, 2007-11-24) McBean, Douglas; Grant, L.; Fyfe, Lorna; Warnock, MaryHarpagophytum procumbens (Hp), commonly known as Devil's Claw is a perennial plant which thrives in arid conditions. For centuries, it has been used as a traditional treatment for a variety of illnesses, including fevers, skin complaints, arthritis and diseases of the digestive tract as well as an appetite stimulant. Since its introduction to Europe in the early twentieth century, it has become a popular antiinflammatory and analgesic preparation amongst herbalists for supportive or adjuvant treatment of degenerative joint diseases, tendonitis, headache, backache and menstrual pain. The validity of Hp as an effective antiinflammatory and analgesic preparation, particularly in the relief of arthritic symptoms, has been investigated in numerous animal, clinical and in vitro studies. Although some contradictory evidence exists, the majority of animal studies appear to indicate Hp as an effective antiinflammatory and analgesic preparation in the treatment of acute and subacute inflammation. Clinical trials support Hp as a beneficial treatment for the alleviation of pain and improvement of mobility in a variety of musculoskeletal conditions. Analysis of the in vitro and ex vivo studies that currently exist, indicate that Hp has significant effects on numerous proinflammatory markers. However, the exact mechanism(s) by which Hp may reduce inflammation remain to be elucidated.Item Pathogenic obesity and nutraceuticals(Cambridge University Press, 2011-08-22) Conroy, Kylie; Davidson, Isobel; Warnock, MaryOver a decade of intense research in the field of obesity has led to the knowledge that chronic, excessive adipose tissue expansion leads to an increase in the risk for CVD, type 2 diabetes mellitus and cancer. This is primarily thought to stem from the low-grade, systemic inflammatory response syndrome that characterises adipose tissue in obesity, and this itself is thought to arise from the complex interplay of factors including metabolic endotoxaemia, increased plasma NEFA, hypertrophic adipocytes and localised hypoxia. Plasma concentrations of vitamins and antioxidants are lower in obese individuals than in the non-obese, which is hypothesised to negatively affect the development of inflammation and disease in obesity. This paper provides a review of the current literature investigating the potential of nutraceuticals to ameliorate the development of oxidative stress and inflammation in obesity, thereby limiting the onset of obesity complications. Research has found nutraceuticals able to positively modulate the activity of adipocyte cell lines and further positive effects have been found in other aspects of pathogenic obesity. While their ability to affect weight loss is still controversial, it is clear that they have a great potential to reverse the development of overweight and obesity-related comorbidities; this, however, still requires much research especially that utilising well-structured randomised controlled trials. 2011 The Authors.Item Physiological levels of soy isoflavones inhibit the growth of oestrogen receptor β-positive but not α-positive breast cancer cells(Society for Endocrinology, 2012-03) Wallace, J.; Gow, Iain F.; Warnock, MaryThe biphasic effect of soy isoflavones (genistein and daidzein) on oestrogen receptor (ER)α+ breast cancer cell proliferation is well documented, with concentrations of ≤10 µM promoting proliferation, and growth inhibitory and apoptotic effects seen at concentrations ≥10 µM. However genistein concentrations as low as 1nM may inhibit the proliferation of some ERα-/β+ cells. This is at odds with epidemiological evidence which suggests that high serum levels of soy isoflavones (around 1 µM) are associated with reduced breast cancer risk, particularly for ERα+ and premenopausal cancers. It is possible that interactions with endogenous oestrogens may be behind this discrepancy. The object here was to investigate combinations of genistein and daidzein at levels achievable in the serum through diet alone, at pre- and postmenopausal oestrogen levels, on breast cancer cell proliferation and apoptosis. ERα+ MCF7 and ERα-/β+ MDA-MB-231 cells were treated with genistein, daidzein and 17β-oestradiol at physiologically relevant levels. Physiological levels of soy isoflavones promoted but not inhibit the proliferation of MCF7 cells, whilst simultaneously inducing low level apoptosis. E2 acted synergistically on proliferation with the isoflavones, increasing proliferation further, but did not impact upon their apoptotic capacity. All concentrations of genistein and daidzein resulted in a slight reduction in MDA-MB-231 proliferation, with this dropping dramatically at the highest dose of 31.6 µM. Overall this suggests that physiological levels of isoflavones may be of benefit as chemotherapeutic agents against ERα-/β+ breast cancer. However they are not capable of reducing the proliferation of ERα+ MCF7 cells, or inhibiting oestrogenic promotion of growth. Other factors must be involved, not reflected by this model. Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.Item Sea Buckthorn (Hippophae rhamnoides L) : A Review and its potential as a Crop in Scotland(2008) Warnock, Mary; Miskin, DavidItem Study of low fat Milk-Based Sports Drink(Robert Wiseman Dairies, 2009) Warnock, Mary; Drummond, SandraItem Anti-microbial activity and composition of Manuka and Portobello honey(John Wiley & Sons, Ltd, 2013-08) Schneider, M.; Coyle, S.; Warnock, Mary; Gow, Iain F.; Fyfe, LornaRecently renewed interest in the therapeutic properties of honey has led to the search for new antimicrobial honeys. This study was undertaken to assess the antimicrobial activity and composition of a locally produced Portobello honey (PBH) on three bacteria known to infect wounds. Manuka honey (MH) was used for comparative purposes. Broth culture and agar disc diffusion assays were used to investigate the antimicrobial properties of honey. The honeys were tested at four concentrations: 75%, 50%, 10% and 1% (v/v) and compared with an untreated control. The composition of honey was determined by measuring: polyphenol content by Folin Ciocalteau method, antioxidant capacity by ferric ion reducing power assay, hydrogen peroxide (H2O2) by catalase test, pH and sugar content by pH strips and refractometer, respectively. Both honeys at 75% and 50% inhibited the majority of the three bacteria tested. 10% PBH exhibited antimicrobial activity to the lesser extent than 10% MH. The difference was very significant (p ≤ 0.001). Both honeys were acidic with pH 4, and both produced H2O2. The sugar content of PBH was higher than MH, but the difference was not significant. The MH had significantly higher levels of the polyphenols and antioxidant activity than PBH. Copyright © 2012 John Wiley & Sons, Ltd.