Sebastian Granica

Role of human gut microbiota metabolism in the anti-inflammatory effect of traditionally used ellagitannin-rich plant materials

ETHNOPHARMACOLOGICAL RELEVANCE Ellagitannin-rich plant materials are widely used in traditional medicine as effective, internally used anti-inflammatory agents. Due to the not well-established bioavailability of ellagitannins, the mechanisms of observed therapeutic effects following oral administration still remain unclear. The aim of the study was to evaluate if selected ellagitannin-rich plant materials could be the source of bioavailable gut microbiota metabolites, i.e. urolithins, together with determination of the anti-inflammatory activity of the metabolites produced on the THP-1 cell line derived macrophages model. MATERIALS AND METHODS The formation of urolithins was determined by ex vivo incubation of human fecal samples with aqueous extracts from selected plant materials. The anti-inflammatory activity study of metabolites was determined on PMA differentiated, IFN-γ and LPS stimulated, human THP-1 cell line-derived macrophages. RESULTS The formation of urolithin A, B and C by human gut microbiota was established for aqueous extracts from Filipendula ulmaria (L.) Maxim. herb (Ph. Eur.), Geranium pratense L. herb, Geranium robertianum L. herb, Geum urbanum L. root and rhizome, Lythrum salicaria L. herb (Ph. Eur.), Potentilla anserina L. herb, Potentilla erecta (L.) Raeusch rhizome (Ph. Eur.), Quercus robur L. bark (Ph. Eur.), Rubus idaeus L. leaf, Rubus fruticosus L. and pure ellagitannin vescalagin. Significant inhibition of TNF-α production was determined for all urolithins, while for the most potent urolithin A inhibition was observed at nanomolar concentrations (at 0.625 μM 29.2±6.4% of inhibition). Urolithin C was the only compound inhibiting IL-6 production (at 0.625 μM 13.9±2.2% of inhibition). CONCLUSIONS The data obtained clearly indicate that in the case of peroral use of the examined ellagitannin-rich plant materials the bioactivity of gut microbiota metabolites, i.e. urolithins, has to be taken under consideration.

Extracts from Epilobium sp. Herbs, Their Components and Gut Microbiota Metabolites of Epilobium Ellagitannins, Urolithins, Inhibit Hormone-Dependent Prostate Cancer Cells-(LNCaP) Proliferation and PSA Secretion

Extracts from Epilobium sp. herbs have been traditionally used in the treatment of prostate‐associated ailments. Our studies demonstrated that the extracts from Epilobium angustifolium, Epilobium parviflorum and Epilobium hirsutum herbs are potent prostate cancer cells (LNCaP) proliferation inhibitors with IC50 values around 35 µg/ml. The tested extracts reduced prostate specific antigen (PSA) secretion (from 325.6 ± 25.3 ng/ml to ~90 ng/ml) and inhibited arginase activity (from 65.2 ± 1.1 mUnits of urea/mg of protein to ~40 mUnits of urea/mg protein). Selected constituents of extracts (oenothein B, quercetin‐3‐O‐glucuronide, myricetin‐3‐O‐rhamnoside) were proven to be active in relation to LNCaP cells. However, oenothein B was the strongest inhibitor of cells proliferation (IC50 = 7.8 ± 0.8 μM), PSA secretion (IC50 = 21.9 ± 3.2 μM) and arginase activity (IC50 = 19.2 ± 2.0 μM). Additionally, ellagitannins from E. hirustum extract were proven to be transformed by human gut microbiota into urolithins. Urolithin C showed the strongest activity in the inhibition of cell proliferation (IC50 = 35.2 ± 3.7 μM), PSA secretion (reduced PSA secretion to the level of 100.7 ± 31.0 ng/ml) and arginase activity (reduced to the level of 27.9 ± 3.3 mUnits of urea/mg of protein). Results of the work offer an explanation of the activity of Epilobium extracts and support the use of Epilobium preparations in the treatment of prostate diseases. Copyright

Development and validation of HPLC-DAD-CAD–MS3 method for qualitative and quantitative standardization of polyphenols in Agrimoniae eupatoriae herba (Ph. Eur)

A reversed phase high performance liquid chromatography method (HPLC) coupled with a diode array, charged aerosol detector or mass spectrometer was developed for the quantitative and qualitative standardization of Agrimoniae eupatoriae herba (Ph. Eur). Twenty four constituents comprising phenolic acids, flavan-3-ol derivatives, ellagitannin and flavonoids were fully or partially identified. Eight of detected compounds were reported from common agrimony for the first time. Fourteen major polyphenols were quantified using validated HPLC method with UV-vis and corona charged detection. Both detectors were shown to be equal for the quantification of selected polyphenols. Some limitations of universal response of corona charged detector for phenolic compounds were discussed. Using obtained data for DAD detector the sum of tannins, flavonoids and phenolic acids quantified in examined plant material was determined. Investigated samples contained 8.2-10.9mg/g of flavonoids, 6.3-10.9mg/g of tannins (among which agrimoniin was dominating constituent 2.6-5.4mg/g) and 0.6-0.9mg/g of phenolic acids proving that flavonoids should be considered as second major group of constituents in common agrimony.

Urolithins, gut microbiota-derived metabolites of ellagitannins, inhibit LPS-induced inflammation in RAW 264.7 murine macrophages.

SCOPE Ellagitannin-rich food products and medicinal plant materials were shown to have beneficial effects toward intestinal inflammation. Due to the questionable bioavailability of ellagitannins their gut microbiota metabolites-urolithins have come to be regarded as potential factors responsible for biological activities observed in vivo. The aim of the study was to determine the influence of the three most abundant bioavailable ellagitannin gut microbiota metabolites-urolithins A, B, and C on inflammatory responses in RAW 264.7 murine macrophages, which are involved in the pathogenesis of intestine inflammation. METHODS AND RESULTS Urolithins A, B, and C decreased NO production via inhibition of the iNOS protein and mRNA expression. They decreased the expression of IL-1β, TNF-α, and IL-6 mRNA in LPS challenged RAW 264.7 murine macrophages. A clear inhibition of NF-κB p65 nuclear translocation and p50 DNA-binding activity was associated with the observed anti-inflammatory activities of urolithins. Among the tested compounds urolithin A had the strongest anti-inflammatory activity. CONCLUSION The anti-inflammatory effects of urolithins at concentrations that are physiologically relevant for gut tissues (≥40 μM), as revealed in this study, support the data from in vivo studies showing the beneficial effects of ellagitannin-rich products toward intestinal inflammation.

Influence of Gut Microbiota-Derived Ellagitanninsʼ Metabolites Urolithins on Pro-Inflammatory Activities of Human Neutrophils

Ellagitannin-rich products exhibit beneficial influence in the case of inflammation-associated diseases. Urolithins, metabolites of ellagitannins produced by gut microbiota, in contrary to high molecular weight hydrophilic parental polyphenols, possess well established bioavailability. Because of the important role of neutrophils in progression of inflammation, the influence of urolithins on their pro-inflammatory functions was tested. Urolithin B at a concentration of 20 µM showed significant inhibition of interleukin 8 and extracellular matrix-degrading enzyme MMP-9 production. It was also significantly active in prevention of cytochalasin A/formyl-met-leu-phenylalanine-triggered selectin CD62L shedding. Urolithin C was the only active compound towards inhibition of elastase release from cytochalasin A/formyl-met-leu-phenylalanine-stimulated neutrophils with 39.0 ± 15.9% inhibition at a concentration of 5 µM. Myeloperoxidase release was inhibited by urolithins A and C (at 20 µM by 46.7 ± 16.1 and 63.8 ± 8.6%, respectively). Urolithin A was the most potent reactive oxygen species release inhibitor both in formyl-met-leu-phenylalanine and 4β-phorbol-12β-myristate-R13-acetate-stimulated neutrophils. At the concentration of 1 µM, it caused reactive oxygen species level decrease by 42.6 ± 26.6 and 53.7 ± 16.0%, respectively. Urolithins can specifically modulate inflammatory functions of neutrophils, and thus could contribute to the beneficial health effects of ellagitannin-rich medicinal plant materials and food products.

Determination of Macrocyclic Ellagitannin Oenothein B in Plant Materials by HPLC‐DAD‐MS: Method Development and Validation

INTRODUCTION Oenothein B (OeB) is a dimeric macrocyclic ellagitannin occurring mainly in the genus Oenothera. In the literature no validated HPLC method for quantification of this compound has been reported. OBJECTIVE To develop and validate an efficient HPLC method for quantification of OeB in plant materials. METHODOLOGY Extraction conditions of OeB from plant material were optimised. Then the first validated HPLC method for quantification was developed. RESULTS We have shown that ultrasonic-assisted extraction at 40 °C in 15 min using water are the most optimal conditions for OeB extraction. We have quantified OeB in several plant materials belonging to Oenotheraceae family. The lowest amount of OeB was present in Circaea lutetiana herb (27.64 ± 0.26 mg/g) and the highest was quantified in Epilobium hirsutum aerial parts (72.91 ± 1.38 mg/g). CONCLUSION The first HPLC method for quantification of OeB in plant materials was developed and validated. We also for the first time optimised the extraction conditions for OeB.

Chemical Composition, Antioxidative and Anti-Inflammatory Activity of Extracts Prepared from Aerial Parts of Oenothera biennis L. and Oenothera paradoxa Hudziok Obtained after Seeds Cultivation

In the present study we investigated the chemical composition of extracts prepared from aerial parts of Oenothera paradoxa Hudziok and Oenothera biennis L. and their antioxidative and anti-inflammatory activities. Ultra high pressure liquid chromatography (UHPLC)-DAD-MS/MS studies showed that both extracts contain a wide variety of polyphenols (39 identified constituents) among which macrocyclic ellagitannin turned out to be the main constituent. During the in vitro studies, using noncellular models, both extracts scavenged reactive oxygen species (ROS) in a concentration-dependent manner, and the lowest SC(50) values were obtained for O(2)(-) and H(2)O(2). Both extracts inhibited ROS production by stimulated human neutrophils. The stronger activity in the case of formyl-met-leu-phenylalanine stimulation suggests that both extracts may act through the receptor-dependent pathway. O. paradoxa extract and O. biennis extract exhibited anti-inflammatory activity by the inhibition of hyaluronidase and lipoxygenase in a concentration-dependent manner. The stronger activity of O.biennis extract toward lipoxygenase may be explained by its higher oenothein B content.

Antioxidant and anti-inflammatory flavonol glucuronides from Polygonum aviculare L.

A series of 11 flavonol glucuronides were isolated from the herb of Polygonum aviculare L. (Ph.Eur) of which 8 were reported for the first time from the Polygonum species. Three acetylated kaempferol and isorhamnetin glucuronides were isolated from a natural source for the first time. All compounds, including the new ones, were characterized using 1D and 2D NMR spectra as well as high resolution mass spectrometry. The influence of all isolated compounds on the production of reactive oxygen species, as well as on elastase release by human neutrophils, was evaluated in in vitro studies. The results showed that all investigated compounds at physiologically achievable concentrations within the range of 0.5-10 μM significantly inhibited the production of reactive oxygen species as well as elastase release in human neutrophils model and should be considered as responsible for anti-inflammatory activity of the P. aviculare herb. The chemotaxonomic value of isolated compounds was also discussed.

Phytochemistry, pharmacology and traditional uses of different Epilobium species (Onagraceae): A review

ETHNOPHARMACOLOGICAL RELEVANCE The Epilobium genus (willowherb) comprises of ca. 200 species of herbaceous plants distributed around the world. Infusions prepared form willowherbs have been traditionally used externally in skin and mucosa infections and in the treatment of benign prostate hyperplasia. Nowadays extracts from different Epilobium species are widely used by patients, however the lack of clinical studies does not allow to fully establish their efficacy. The present review summarizes published data on phytochemistry, ethnopharmacological use and pharmacological studies concerning willowherb species investigated throughout past few decades. MATERIALS AND METHODS Literature survey was performed using Scopus, PubMed, Web of Science and Reaxys databases looking for papers and patents focused on chemical composition and bioactivity of Epilobium species. Systematic research in ethnopharmacological literature in digitalized sources of academic libraries was also carried out. RESULT The chemical composition of different Epilobium species and their bioactivities are described. The detailed information on constituents isolated and detected by chromatographic methods is given. The studies show that polyphenols are main compounds occurring in Epilobium herb among which flavonoids, phenolic acids and tannins (oenothein B and oenothein A) are dominating constituents. The extracts and some isolated compounds from Epilobium sp. were shown to possess antimicrobial, anti-proliferative, anti-inflammatory, analgesic and antioxidative activities. Because many studies suggest that oenothein B as dominating constituent may be responsible for Epilobium sp. pharmacological effects, its documented bioactivities were also described. CONCLUSIONS The pharmacological studies performed on Epilobium justify the traditional use of this species in external and in gastrointestinal inflammations. As far as the treatment of benign prostate hyperplasia (BPH) is considered, in the literature, there are some reports indicating that Epilobium extracts have a beneficial effect for this disorder, but the number of in vitro studies is not sufficient and the in vivo studies are not conclusive or too preliminary to draw a final conclusion about the efficacy of Epilobium preparations. More in vitro, in vivo and clinical studies to confirm this mode of action are strongly needed. Epilobiums extracts have also documented antioxidative and potential anti-inflammatory properties. Oenothein B can be considered as responsible for some of Epilobium pharmacological properties. Because of the lack of clinical data further studies are needed to provide an evidence base for traditional uses of plant materials belonging to the Epilobium genus.

Polyphenolic Profile, Antioxidant and Anti-Inflammatory Activity of Eastern Teaberry (Gaultheria procumbens L.) Leaf Extracts.

Dry leaf extracts of eastern teaberry (Gaultheria procumbens L.) were evaluated as a source of bioactive phytocompounds through systematic activity testing and phytochemical profiling. The antioxidant efficiency was tested using five complementary in vitro models (DPPH; FRAP; linoleic acid (LA) peroxidation assay; O2•− and H2O2 scavenging tests) in parallel with standard antioxidants. The 75% methanol extract and its diethyl ether, ethyl acetate (EAF), n-butanol and water fractions exhibited the dose-dependent responses in all assays, with the highest capacities found for EAF (DPPH EC50 = 2.9 μg/mL; FRAP = 12.8 mmol Fe2+/g; IC50 for LA-peroxidation = 123.9 μg/mL; O2•− SC50 = 3.9 μg/mL; H2O2 SC50 = 7.2 μg/mL). The EAF had also the highest anti-inflammatory activity in the inhibition tests of lipoxygenase and hyaluronidase (60.14% and 21.83% effects, respectively, at the concentration of 100 μg/mL). Activity parameters of the extracts correlated strongly with the levels of total phenolics (72.4–270.7 mg GAE/g), procyanidins, and phenolic acids, whereas for flavonoids only moderate effects were observed. Comprehensive UHPLC-PDA-ESI-MS3 and HPLC-PDA studies led to the identification of 35 polyphenols with a procyanidin A-type trimer, quercetin 3-O-glucuronide, isomers of caffeoylquinic acids, and (‒)-epicatechin being the dominant components. Significant activity levels, high phenolic contents and high extraction yields (39.4%–42.5% DW for defatted and crude methanol extracts, respectively) indicate the value of eastern teaberry leaves as bioactive products.