21 research outputs found
FT-IR Method for the Quantification of Isoflavonol Glycosides in Nutritional Supplements of Soy (Glycine max (L.) MERR.)
Due to increasing health consciousness, a lot of food supplements are sold and
used. Dietary supplements of Glycine max (L.) MERR. are used as an alternative
treatment for menopausal complaints such as hot flashes. Thereby, the
effective soy compounds are the isoflavones daidzin, genistin, and glycitin.
However, only the total soy extract content of the nutritional supplements is
indicated. The aim of this study is to introduce a fast, efficient, and
economic Fourier transformation infrared (FT-IR) spectroscopy method to
quantify the active ingredients in the complex matrix of soy-based
supplements. Five different nutritional supplements of Glycine max (L.) MERR.
were purchased from a German pharmacy and were extracted with 80% aqueous
methanol. A high-performance liquid chromatography (HPLC) method was used for
the separation. The samples were concentrated and measured with infrared
spectroscopy. An FT-IR method was established to quantify the active
ingredients in the complex matrix of soy-based nutritional supplements. The
partial least-squares algorithm was used to develop the method, which enabled
the estimation of the content of particular isoflavones (daidzin R² = 0.86,
glycitin R² = 0.94, genistin R² = 0.96) and the quantification of the total
isoflavone content (R² = 0.92) despite peak overlap in the infrared (IR)
spectra. The method for the quantification of the isoflavonol glycosides is
precise with the standard error of prediction being 13.54%
Impact of Mistletoe Triterpene Acids on the Uptake of Mistletoe Lectin by Cultured Tumor Cells
Complementary treatment possibilities for the therapy of cancer are increasing
in demand due to the severe side effects of the standard cytostatics used in
the first-line therapy. A common approach as a complementary treatment is the
use of aqueous extracts of Viscum album L. (Santalaceace). The therapeutic
activity of these extracts is attributed to Mistletoe lectins which are
Ribosome-inactivating proteins type II. Besides these main constituents the
extract of Viscum album L. comprises also a mixture of lipophilic ingredients
like triterpene acids of the oleanane, lupane and ursane type. However, these
constituents are not contained in commercially available aqueous extracts due
to their high lipophilicity and insolubility in aqueous extraction media. To
understand the impact of the extract ingredients in cancer therapy, the
intracellular uptake of the mistletoe lectin I (ML) by cultured tumor cells
was investigated in relation to the mistletoe triterpene acids, mainly
oleanolic acid. Firstly, these hydrophobic triterpene acids were solubilized
using cyclodextrins ("TT" extract). Afterwards, the uptake of either single
compounds (isolated ML and the aqueous "viscum" extract) or in combination
with the TT extract (ML+TT, viscumTT), was analyzed. The uptake of ML was
studied inTHP-1-, HL-60-, 143B- and Ewing TC-71-cells and determined after 30,
60 and 120 minutes by an enzyme linked immunosorbent assay which quantifies
the A-chain of the hololectin. It could be shown that the intracellular uptake
after 120 minutes amounted to 20 % in all cell lines after incubation with
viscumTT. The studies further revealed that the uptake in THP-1-, HL-60- and
Ewing TC-71-cells was independent of the addition of TT extract.
Interestingly, the uptake of ML by 143B-cells could only be measured after
addition of triterpenes pointing to resistance to mistletoe lectin
A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo
Aqueous Viscum album L. extracts are widely used in complementary cancer
medicine. Hydrophobic triterpene acids also possess anti-cancer properties,
but due to their low solubility they do not occur in significant amounts in
aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes
(mainly oleanolic acid) and investigated the effect of a mistletoe whole plant
extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo.
Single Viscum album L. extracts containing only solubilised triterpene acids
(TT) or lectins (viscum) inhibited cell proliferation and induced apoptosis in
a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT
extracts (viscumTT) enhanced the induction of apoptosis synergistically. The
experiments demonstrated that all three extracts are able to induce apoptosis
via caspase-8 and -9 dependent pathways with down-regulation of members of the
inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute
myeloid leukaemia mouse model experiment confirmed the therapeutic
effectiveness of viscumTT-treatment resulting in significant tumour weight
reduction, comparable to the effect in cytarabine-treated mice. These results
suggest that the combination viscumTT may have a potential therapeutic value
for the treatment AML
FT-IR Method for the Quantification of Isoflavonol Glycosides in Nutritional Supplements of Soy (Glycine max (L.) MERR.)
Due to increasing health consciousness, a lot of food supplements are sold and used. Dietary supplements of Glycine max (L.) MERR. are used as an alternative treatment for menopausal complaints such as hot flashes. Thereby, the effective soy compounds are the isoflavones daidzin, genistin, and glycitin. However, only the total soy extract content of the nutritional supplements is indicated. The aim of this study is to introduce a fast, efficient, and economic Fourier transformation infrared (FT-IR) spectroscopy method to quantify the active ingredients in the complex matrix of soy-based supplements. Five different nutritional supplements of Glycine max (L.) MERR. were purchased from a German pharmacy and were extracted with 80% aqueous methanol. A high-performance liquid chromatography (HPLC) method was used for the separation. The samples were concentrated and measured with infrared spectroscopy. An FT-IR method was established to quantify the active ingredients in the complex matrix of soy-based nutritional supplements. The partial least-squares algorithm was used to develop the method, which enabled the estimation of the content of particular isoflavones (daidzin R² = 0.86, glycitin R² = 0.94, genistin R² = 0.96) and the quantification of the total isoflavone content (R² = 0.92) despite peak overlap in the infrared (IR) spectra. The method for the quantification of the isoflavonol glycosides is precise with the standard error of prediction being 13.54%
Cell biological and analytical studies on mistletoe lectins and triterpenes (Viscum album L.)
Die Nachfrage nach alternativen Behandlungsmöglichkeiten für verschiedenste
Krebserkrankungen steigt aufgrund der vielfachen Nebenwirkungen der
klassischen Therapie stetig an. Wässrige Mistelpräparate (Viscum album L.
(Santalaceae)) werden bereits vielfach als komplementäre Behandlungsmethode
eingesetzt. Zu den Hauptinhaltsstoffen zählen die Mistellektine, die zu der
Gruppe der Ribosomeninaktivierenden Proteine Typ-II gehören und aufgrund ihrer
N-Glykosidase-Aktivität das hauptsächlich wirksame zytotoxische Prinzip der
Anwendung darstellen. Des Weiteren sind zahlreiche lipophile Substanzen in der
Mistel vorhanden, von denen vor allem die Triterpensäuren, hauptsächlich
Oleanolsäure, aufgrund ihrer antitumoralen und antiinflammatorischen
Eigenschaften von besonderem Interesse sind. Eine Kombination beider
Stoffklassen könnte dementsprechend eine Bereicherung auf den
Arzneimittelmarkt im Bereich der adjuvanten Tumortherapie sein. Die
Quantifizierung der Mistellektine konnte mithilfe zweier etablierter ELISA-
Methoden sowohl im wässrigen als auch im serumhaltigen Medium erfolgen. Die
Triterpensäuren, vor allem Oleanolsäure, konnten mit einer validierten GC /
FID-Methode ebenfalls im lösungsmittel- und im serumhaltigen Milieu bestimmt
werden. Der A-Ketten-ELISA ermöglichte zudem die Ermittlung der
intrazellulären Mistellektin-Konzentration in Leukämie- und Sarkom-Zellen.
Eine Aufnahme von Mistellektinen wurde in jeder kultivierten Tumorzelllinie
detektiert. Bei der Osteosarkomzelllinie 143B konnte jedoch eine Mistellektin-
Aufnahme erst durch den Zusatz des Triterpensäure-haltigen Extraktes
nachgewiesen werden. Eine unterschiedliche Konzentration an Oleanolsäure
spielte bezĂĽglich der Mistellektinaufnahme jedoch keine Rolle. Die kombinierte
Gabe der hydrophilen und lipophilen Stoffgruppen wurde durch den Zusatz von
2-Hydroxypropyl-β-Cyclodextrin ermöglicht, der für die Solubilisierung der
stark lipophilen Triterpensäuren verantwortlich ist. Eine methanolische Lösung
des Triterpensäuren-Extraktes lieferte das gleiche Ergebnis wie der
solubilisierte Triterpensäure-Extrakt. Die Formulierung als Liposomen
(verkapselter Triterpenextrakt) ermöglichte zwar auch eine Aufnahme der
Mistellektine, die aber deutlich geringer ausfiel als der Effekt des in
Methanol gelösten bzw. mit 2-Hydroxypropyl- β-Cyclodextrin solubilisierten
Triterpensäure-Extrakts. Die Formulierung von Mistellektinen in Liposomen
bewirkte eine geringere Aufnahme durch die kultivierten Tumorzellen, die
allerdings rezeptor-unabhängig verläuft. Zusätzlich konnte festgestellt
werden, dass bei der Gabe des wässrigen Viscum-Extraktes ein höherer Anteil an
Mistellektin aufgenommen wurde als bei der Gabe des Mistellektin-Standards.
Bei den Untersuchungen bezüglich der Zytotoxizität konnte mithilfe sowohl
eines Endpunktassays als auch einer Echtzeitanalyse mittels Impedanzmessung
der wässrige Viscum-Extrakt als toxischer eingestuft werden als der
Mistellektin-Standard. Die jeweiligen Kombinationen mit dem Triterpensäure-
haltigen Extrakt führten jedoch schließlich zu keiner signifikant erhöhten
Zytotoxizität, verglichen mit den Einzelkomponenten. Die Verkapselungen der
Einzelkomponenten waren ebenfalls nicht stärker zytotoxisch. Mithilfe der
konfokalen Laser-Scanning-Mikroskopie wurde der Verlauf von
fluoreszenzmarkiertem Mistellektin-Standard innerhalb der Zelle verdeutlicht,
die Lokalisation in Endosomen, Vesikelverschmelzungen und Bläschenbildung in
der Zelle wurden beobachtet. Die Lyse von Vesikeln durch den Zusatz von
triterpensäurehaltigen Extrakten konnte punktuell hervorgerufen und beobachtet
werden.Complementary treatment possibilities for cancer diseases are increasing in
demand due to the severe side effects of the standard first-line therapy. A
common approach as a complementary treatment is the use of aqueous extracts of
Viscum album L. (Santalaceae). The therapeutic activity of the extracts is
attributed to the N-glycosidase-activity of the mistletoe lectins which are
ribosome-inactivating proteins type II. Besides these constituents, the
spectrum of Viscum album L. comprises also a mixture of lipophilic
ingredients, mainly triterpene acids, like oleanolic acid which have antitumor
and antiinflammatory effects. The combined application of both substance
classes could be an enrichment for the adjuvant chemotherapy. The mistletoe
lectins could be quantified by two ELISAs in aqueous and in serum containing
media. A validated GC / FID-method was used to quantify the triterpene acids,
especially oleanolic acid, in organic solvents and also in serum containing
media. The intracellular uptake of the mistletoe lectin by leukemia and
sarcoma cell lines was analyzed by the A-chain-ELISA. The uptake of mistletoe
lectins was determined in all cultured tumor cells. For the 143B osteosarcoma
cell line, only the addition of triterpene acids resulted in a measurable
intracellular mistletoe lectin concentration. The examination of the impact of
the triterpene acid concentration (calculated as oleanolic acid) on the uptake
of mistletoe lectin has shown no higher uptake with increasing triterpene acid
concentration. The combined application of the hydrophilic and lipophilic
substance classes becomes feasible by solubilization of the triterpene acids
using 2-hydroxypropyl-β-cyclodextrin. The addition of a nonsolubilized
lipophilic extract (without 2-hydroxypropyl-β-cyclodextrin) resulted in the
same intracellular mistletoe lectin concentration. The modeling as liposomes
was enabled a smaller mistletoe lectin uptake than by using the triterpene
acid containing extract in methanol or with 2-hydroxypropyl-β-cyclodextrin.
The uptake of mistletoe lectin I from liposomes was also limited. Furthermore,
the mistletoe lectin uptake from the viscum extracts by the cells was higher
than the uptake of the isolated mistletoe lectin. The examinations of the
cytotoxicity using endpoint determinations and real-time analysis has also
shown a higher toxicity of the viscum extracts in comparison to the mistletoe
lectin standard. The addition of the triterpene acid containing extract or the
application as liposomes did not significantly enhance the cytotoxixity. Using
confocal laser scanning microscopy, the intracellular transport of
fluorescence labeled mistletoe lectin could be analyzed. Vesiculations,
localization in endosomes and fusions of vesicles were recognized. The lysis
of vesicles could be selectively observed after addition of triterpene acids
Used isolated ML and isolated ML + OA concentrations.
<p>Used isolated ML and isolated ML + OA concentrations.</p
Comparison between TT extract solubilized with 2-HP-β-CD and solved in MeOH.
<p>Comparison between TT extract solubilized with 2-HP-β-CD and solved in MeOH.</p
Cell viability of HL-60-cells.
<p>The cells were treated with different ML concentrations (see Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153825#pone.0153825.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153825#pone.0153825.t003" target="_blank">3</a>) for 30, 60 and 120 minutes. The isolated ML, three viscum extract batches and the viscum extract batch 161 in combination with TT 161 extract batch (25 ÎĽg/mL and 35 ÎĽg/mL OA) were used. The viability was determined with Annexin V-APC and propidium iodide by flow cytometry. The values are expressed as percentages of the untreated control cells. Error bars represent the standard deviation of n = 2 experiments.</p
Comparison between 2-HP-β-CD and TT extract.
<p>Comparison between 2-HP-β-CD and TT extract.</p