Comparative study on saponin contents of Asparagus officinalis

In der vorliegenden Arbeit wurde der Saponingehalt von in Deutschland angebautem Spargel bestimmt. Dabei wurde auch betrachtet, welche Faktoren vor und nach der Ernte Einfluss auf diese Gehalte haben. Saponine sind wegen zahlreicher biologischer Aktivitäten (wie z.B. antimikrobieller Eigenschaften) von wissenschaftlichem Interesse. Für die Untersuchungen stand Probenmaterial aus einer Gewächshausanlage des IGZ sowie aus dem Brandenburger Sortenversuch zur Verfügung. Die Thermostabilität von Saponinen wurde einerseits an Saponinextrakten bei unterschiedlicher Temperatureinwirkung und andererseits an gefriergelagertem Spargel untersucht. Die Probenaufbereitung erfolgte mittels Festphasenextraktion (SPE). Die qualitative und quantitative Analytik wurde mittels Dünnschichtchromatographie (DC) vorgenommen. Es wurden Gesamtsaponingehalte zwischen ca. 14 mg bis 80 mg in 100 g Frischmasse ermittelt. Dabei zeigten sich eindeutig sortenabhängige Unterschiede sowie eine unterschiedliche Reaktion auf solche Faktoren wie Bodentemperatur, möglicherweise Bodenfeuchte und pflanzenspezifische Aspekte, wie z.B. einem Befall mit Pathogenen. Während Erhitzen von Spargelextrakten zu temperaturabhängigen Saponinverlusten führte, wiesen gefriergelagerte Spargelproben nach 12 Wochen keine Saponinverluste auf. In Auswertung der Ergebnisse scheint eine Aufbereitung von Spargelabfällen zur Gewinnung von Reinsubstanzen vor dem Hintergrund, dass in Deutschland pro Erntesaison ca. 25.000 Tonnen dieser Abfälle entstehen, als sinnvoll. Für den Einsatz dieser aufgereinigten Saponine sind verschiedene Anwendungsgebiete denkbar. Dazu gehören funktionelle Lebensmittel, Nutraceuticals und biologische Pflanzenschutzmittel.In this work we estimated the quantity of saponins in asparagus spears, grown in Germany, with respect to different cultivars as well as pre and post harvest treatments. Saponins are known as biologically active substances. For our investigation we used plant material grown in the greenhouse of the IGZ Großbeeren as well as in cultivar experiment in Brandenburg. A method was developed to prepare samples by Solid Phase Extraction (SPE) and to analyse these extracts by High Performance Thin Layer Chromatography (HPTLC). With this method contents of total saponins of approximately 14 mg to 80 mg per 100 g fresh weight were measured. Significant differences between cultivars were found. But other pre harvest conditions like low soil moisture or pathogens attacks may also have effected the contents of saponins. Heating of Asparagus extracts caused losses of saponins, depending on the increasing of temperature. No losses of saponins were detected in frozen asparagus samples after 12 weeks of storage. About 25.000 t of broken asparagus spears, bottom cuts and other unmarketable parts are disposed every season in Germany and mainly they are biologically degradated. The interpretation of our results suggests that it may be useful to process these waste products, to clean up saponins and to use these prepared saponins as additives to functional food, nutraceuticals or natural pesticides

Age-related differences in skeletal muscle microvascular response to exercise as detected by contrast-enhanced ultrasound (CEUS).

BACKGROUND:Aging involves reductions in exercise total limb blood flow and exercise capacity. We hypothesized that this may involve early age-related impairments of skeletal muscle microvascular responsiveness as previously reported for insulin but not for exercise stimuli in humans. METHODS:Using an isometric exercise model, we studied the effect of age on contrast-enhanced ultrasound (CEUS) parameters, i.e. microvascular blood volume (MBV), flow velocity (MFV) and blood flow (MBF) calculated from replenishment of Sonovue contrast-agent microbubbles after their destruction. CEUS was applied to the vastus lateralis (VLat) and intermedius (VInt) muscle in 15 middle-aged (MA, 43.6±1.5 years) and 11 young (YG, 24.1±0.6 years) healthy males before, during, and after 2 min of isometric knee extension at 15% of peak torque (PT). In addition, total leg blood flow as recorded by femoral artery Doppler-flow. Moreover, fiber-type-specific and overall capillarisation as well as fiber composition were additionally assessed in Vlat biopsies obtained from CEUS site. MA and YG had similar quadriceps muscle MRT-volume or PT and maximal oxygen uptake as well as a normal cardiovascular risk factors and intima-media-thickness. RESULTS:During isometric exercise MA compared to YG reached significantly lower levels in MFV (0.123±0.016 vs. 0.208±0.036 a.u.) and MBF (0.007±0.001 vs. 0.012±0.002 a.u.). In the VInt the (post-occlusive hyperemia) post-exercise peaks in MBV and MBF were significantly lower in MA vs. YG. Capillary density, capillary fiber contacts and femoral artery Doppler were similar between MA and YG. CONCLUSIONS:In the absence of significant age-related reductions in capillarisation, total leg blood flow or muscle mass, healthy middle-aged males reveal impaired skeletal muscle microcirculatory responses to isometric exercise. Whether this limits isometric muscle performance remains to be assessed

‘Experimental protocol’.

<p>Fig 1a) Study protocol with time schedule for exercise, Sonovue infusion, CEUS recordings, as well as femoral artery Doppler and brachial blood pressure measurements at rest, during isometric exercise and during post-exercise hyperemia. The time points of high-MI US-destruction of the Sonovue microbubbles are indicated by arrows, each of which was followed by a low-MI recording of Sonovue replenishment curves covering 25 s. Fig 1b) Example of a torque recording during isometric knee extension as controlled by the subject through visual feed-back.</p

‘Total leg blood flow and conductance’.

<p>Mean (±SEM) total leg blood flow (femoral artery Duplex-Doppler flow), calculated total leg vascular conductance (leg blood flow per mean arterial pressure), and systolic as well as diastolic brachial arterial blood pressure at rest in middle-aged (MA, n = 15) compared to young (YG, n = 11) males after ~90 s of isometric exercise and ~60 s post-exercise. # for p<0.05 by unpaired Student’s t-test middle-aged MA vs. YG. * for p<0.05, ** for p<0.01, and *** for p<0.001 by paired Student’s t-test for changes relative to rest (baseline) within the group of MA or YG.</p

‘Ultrasound and MRT imaging of CEUS and Biopsy muscle site’.

<p>Fig 2 a) US B-mode image of a typical combined vastus lateralis (VLat) and intermedius (VInt) CEUS scan with the scanner position chosen parallel to the VLat muscle fiber orientation (i.e. from proximal/lateral to distal/medial). The intramuscular septum separating both muscles is indicated, the mean depth was similar between middle aged (MA) and young (YG) subjects and not significantly different between the conditions before, during, and post-exercise (see also the ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172771#sec006" target="_blank">Methods</a>‘ section on Contrast-enhances US (CEUS). d) Thigh MRT-imaging transversal section at the exact site of VLat muscle biopsy (<i>middle</i>) and CEUS recording as well as 1 cm proximal (<i>left</i>) and distal (<i>right</i>). Note that this MRT was obtained 3 h after a muscle biopsy to visualize the exact biopsy site (local fluid /blood accumulation) indicated by the arrow.</p

‘Means of measured replenishment curves and individual regression lines’.

<p>Fig 4a) Mean replenishment curves (RC) in the vastus lateralis (VLat; <i>upper panel</i>) and the vastus intemedius (VInt; <i>lower panel</i>) muscle ROI of middle-aged (MA, n = 15) compared to young (YG, n = 11) males during rest (left), after 70 s isometric exercise (middle), and 15 s post-exercise. Note differences in initial slope or in the plateau reached during or post-exercise. Fig 4b) Mean regression lines, corresponding to mean RC presented above in a) i.e. for the VLat (<i>upper panel</i>) and the VInt (<i>lower panel</i>) muscle ROI of middle-aged (MA, n = 15) compared to young (YG, n = 11) males during rest (left), after 70 s isometric exercise (middle), and 15 s post-exercise. Note the differences in initial slope or reached plateau during or post exercise. Furthermore, note that regression lines represent the mean of individual regression lines calculated for individual RCs (not the regression line calculated for mean RCs, presented above in a). For statistical differences between MA and YG regarding the RC-derived parameters of microvascular blood volume (MBV), flow velocity (MFV), and blood flow (MBF) please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172771#pone.0172771.g005" target="_blank">Fig 5</a>.</p

‘Time courses of the mean contrast-agent CEUS signals’.

<p>Time course of the mean (±SEM) Sonovue microbubble signal in the vastus lateralis (VLat; <i>upper panel</i>) and the vastus intermedius (VInt; <i>lower panel</i>) muscle region of interests (ROI) in middle-aged (MA, n = 15) and young (YG, n = 11) males in the experimental intervals: equilibration at rest (<i>left;</i> initial 180 s of Sonovue infusion), isometric exercise (<i>middle</i>; first 60 s of knee extension at 15% of PT), and post-exercise (<i>right;</i> initial 15 s after cessation of). Note the different time scales on the x-axis with these three conditions. The time intervals for repetitive Sonovue replenishment curve (RC) recording following high-MI US destruction of Sonovue microbubbles (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172771#pone.0172771.g001" target="_blank">Fig 1</a>) are presented separately in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172771#pone.0172771.g004" target="_blank">Fig 4A</a> (mean RC curves) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172771#pone.0172771.g004" target="_blank">Fig 4B</a> (means of the individual regression lines obtained from individual RC curves). * for p<0.05 MA vs YG by unpaired Student’s t-test.</p

‘Microvascular blood volume (MBV), flow velocity (MFV) and blood flow (MBF)’.

<p>Mean (±SEM) microvascular blood volume (MBV, <i>left</i>), flow velocity (MFV, <i>middle</i>), and blood flow (MBF, <i>right</i>) in the vastus lateralis (VLat; <i>upper panel</i>) and the vastus intemedius (VInt; <i>lower panel</i>) muscle ROI of middle-aged (MA, n = 15) compared to young (YG, n = 11) at rest (two measurements), after 70 and 95 s of isometric exercise and 15, 30 60 and 90 s post-exercise. Note that these data were individually calculated from individual RC curve regression before averaging them for MA or YG (for mean RCs and regression lines per group see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172771#pone.0172771.g004" target="_blank">Fig 4A</a>). # for p<0.05 by unpaired Student’s t-test middle-aged MA vs. YG. * for p<0.05, ** for p<0.01, and *** for p<0.001 by paired Student’s t-test for changes relative to rest (basline) within the group of MA or YG.</p