The absorption of xanthohumol in in vitro and in vivo studies and the investigation of the biological activity of structurally related chalcones

URN to cite this document:: urn:nbn:de:bvb:355-epub-265720
DOI to cite this document:: 10.5283/epub.26572

Motyl, Magdalena

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Date of publication of this fulltext: 16 Jan 2013 07:47

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Item type:	Thesis of the University of Regensburg (PhD)
Open Access Type:	Primary Publication
Date:	16 January 2013
Referee:	Prof. Dr. Jörg Heilmann
Date of exam:	26 October 2012
Institutions:	Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical Biology (Prof. Heilmann)
Keywords:	Xanthohumol, Chalcone, absorption, in vivo studies, in vitro studies
Dewey Decimal Classification:	500 Science > 500 Natural sciences & mathematics 500 Science > 540 Chemistry & allied sciences
Status:	Published
Refereed:	Yes, this version has been refereed
Created at the University of Regensburg:	Yes
Item ID:	26572

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Abstract (English)

Abstract (English)

XN is an abundant chalcone in hops and spread a wide range of pharmacological effects. The lipophilicity of XN is a challenge for water based in vitro studies. For this reason suitable experimental set ups for in vitro experiments were established. A content of 10% FCS in the cell culture medium was found to efficiently increase the solubility of XN. However the application of 10% FCS did not fully prevent the adsorption of XN to several cell culture materials. In Greiner culture flasks, TPP culture flasks and Petri dishes but not in centrifuge tubes and 96 well plates a solubility concentration of 10 µM XN could be reached.
XN is mainly found in beer in the human diet and is ingested with other minor hop compounds. After ingestion XN is highly metabolized (see Chapter 5). For this reason it is necessary to determine the effects of minor hop chalcones, XN metabolites and related compounds in order to investigate if the compounds are more or less active compared to XN. XN is a potent anti inflammatory compound that leads to the inhibition of inflammatory and angiogenetic factors and exhibits anti cancer effects. The compounds were investigated concerning their ability to influence the viability, proliferation and cytotoxicity in MTT and CV assays.
Compound 2 had the lowest IC50 values in the MTT assay. In the CV assay 6 and 7 showed the lowest IC50 values in most of the investigated cell lines. Thus these XN metabolites and related compounds might be candidates for advanced studies.
The ability to influence inflammatory factors such as the NO production and iNOS expression was determined in a Griess assay and an iNOS expression assay. Compound 10 effectively inhibited the NO production in RAW 264.7 cells stimulated with 10 ng/ml LPS. The iNOS expression was significantly inhibited by the tested hydrogenated chalcones. In the group of the chalcones the tetrahydropyran chalcone 6 had a remarkable potential to inhibit the NO production and iNOS expression. The non prenylated chalcones 13 and 14 showed a higher activity to inhibit the NO production with both stimuli compared to the prenylated chalcones. In the group of the non prenylated chalcones the chalcones with one hydroxyl group led to more effective inhibition of the NO production and iNOS expression compared to those with two or no hydroxyl groups. In the group of the prenylated chalcones 2 showed the highest and 5 the lowest activity in inhibiting the NO production (stimulus 10 ng/ml LPS) and iNOS expression. Prenylated chalcones with free hydroxyl groups inhibited the NO production more efficient than the O methoxylated and O acetylated derivatives.
Compounds 2, 4, 6 and 7 differ in their activity in the performed assays and might be candidates for the study of molecular mechanism in more detail.
XN exhibits a low bioavailability. The exact uptake mechanism of XN is not fully understood yet. In this thesis the XN uptake was studied in hepatic and colorectal cancer cell lines and primary hepatocytes. HSC, Caco 2 and PH cells exhibited a 52, 63 and 47 fold higher XN concentration compared to the medium which was initially supplemented with 10 µM XN. In HuH 7 cells the measured XN concentration was only 3 fold higher. This might be explained by differences in the ABC transporters, metabolism and amount of proteins per cell. But the reason is not known yet and needs to be further investigated. The results showed that XN is accumulated in hepatic cancer cell lines and primary hepatocytes and might thus exhibit a low bioavailability. This prompted us to investigate the bioavailability as well as the distribution and metabolism of XN in the liver, serum, bile, feces and urine of BALB/c mice.
The BALB/c mice were fed with 100 mg XN/kg, 500 mg XN/kg and 1000 mg XN/kg body weight (73% XN) for 3 and 7 days respectively. There was no linear correlation between the fed amount of XN and the XN concentration in serum. Thus it seems advisable to determine suitable concentration ranges for every compound prior to in vivo experiments. The distribution measurements revealed that the highest XN concentrations were detected in the bile followed by the feces, urine, liver and serum. This pointed to an entero hepatic circulation of XN. The serum XN (unmetabolized) concentration was between 0.01 and 0.12 µM. XN was highly metabolized. The ratio between conjugated and unconjugated XN was between 21 and 163.
In order to investigate the influence of the matrix on the XN absorption, BALB/c mice were fed with 73% XN (27% matrix) and 98% XN. The BALB/c mice received a diet with 1000 mg XN/kg body weight for 3 and 7 days respectively. The absorption of XN was significantly higher in the group that was fed with 73% XN. In addition the studies showed that XN is highly metabolized. The highest XN concentrations were detected in the bile followed by the serum, liver, urine and feces.

Translation of the abstract (German)

Xanthohumol (XN) gehört zu der Gruppe der Chalkone und ist ein zentraler Bestandteil im Hopfen. XN hat vielfältige pharmakologische Effekte. Die Lipophilie von XN ist eine Herausforderung für in vitro Studien in wässrigen Systemen. Aus diesem Grund wurde ein Schwerpunkt dieser Arbeit auf die Entwicklung geeigneter analytischer Verfahren gelegt. Es konnte gezeigt werden, dass der Zusatz von 10 % FCS zu den verwendeten Zellkulturmedien die Löslichkeit von XN erhöht. Allerdings konnte ein Zusatz von 10 % FCS die Adsorption von XN an Zellkulturmaterialien nicht vollständig verhindern. Bei Greiner Kulturflaschen, TPP Kulturflaschen und Petrischalen nicht aber bei Zentrifugenröhrchen und 96-well-Platten konnte eine Löslichkeitskonzentration von bis zu 10 µM XN erreicht werden.
Hopfenbestandteile und XN werden in der täglichen Nahrung in Form von Bier aufgenommen, und im Körper metabolisiert. Aus diesem Grund ist es notwendig bei der Betrachtung der XN Wirkung ebenso dessen Metabolite und Chalkone aus dem Hopfen zu untersuchen. XN weißt anti-inflammatorische, -angiogenetische Wirkung und anti-tumor Eigenschaften auf. Es wurde der Einfluss des XNs, XN Metaboliten und struktur ähnlichen Verbindungen auf den Viabilitäts-, Proliferationseinfluss und deren Zytotoxizität in MTT- und Kristallviolett Assays bestimmt.
Verbindung 2 hatte den geringsten IC50 Wert im MTT-Assay. Für einen Großteil der untersuchten Zelllinien, die Verbindungen 6 und 7 die geringsten IC50 Werte im Kristallviolett Assay. Daher könnten diese Verbindungen geeignete Kandidaten für weitere Untersuchungen sein. Die Fähigkeit der Testsubstanzen pro-inflammatorische Faktoren wie bspw. die NO Produktion und die iNOS Expression zu beeinflussen wurde im Griess-Assay und im iNOS Expresssions-Assay untersucht. Verbindung 10 inhibierte die NO Produktion in RAW 264.7 Zellen, die mit 10 ng/ml LPS stimuliert wurden. Zudem wurde die iNOS Expression signifikant durch hydrierte Chalkone inhibiert. Aus der Gruppe der Chalkone zeigte das Tetrahydropyran-Chalkon 6 ein hohes Potential zur Inhibierung der NO Produktion und der iNOS Expression. Die nicht-prenylierten Chalkone 13 und 14 zeigten eine höhere Inhibierung der NO Produktion verglichen mit den prenylierten-Chalkonen unabhängig von den zwei verwendeten Stimuli. In der Gruppe der nicht-prenylierten Chalkone führten die Chalkone mit einer Hydroxylgruppe zu einer stärkeren Inhibierung der NO Produktion und der iNOS Expression als Chalkone mit zwei oder keinen Hydroxylgruppen. In der Gruppe der prenylierten Chalkone zeigte Verbindung 2 die stärkste Inhibierung und Verbindung 5 die schwächste Inhibierung der NO-Produktion (Stimulus 10 ng/ml LPS) und der iNOS Expression. Prenylierte Chalkone mit freien Hydroxylgruppen inhibierten die NO-Produktion effizienter als O-methoxylierte and O-acetylierte Derivate. Die Verbindungen 2, 4, 6, und 7 unterschieden sich in ihrer Aktivität in den durchgeführten Assays und könnten Kandidaten für weitere Untersuchungen sein.

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