Anna K. Kiss

Anti-hyaluronidase and anti-elastase activity screening of tannin-rich plant materials used in traditional Polish medicine for external treatment of diseases with inflammatory background

ETHNOPHARMACOLOGICAL RELEVANCE The aim of the study was to examine extracts from twelve tannin-rich plant materials used in traditional Polish medicine for external treatment of skin and mucosa diseases considering their ability to inhibit hyaluronidase activity and elastase release from stimulated neutrophils in vitro. MATERIALS AND METHODS In vitro anti-hyaluronidase and anti-elastase assays together with phytochemical qualitative and quantitative screening were performed. RESULTS The strongest inhibition of hyaluronidase was observed for extract from Lythri herba, with IC(50) value 8.1 ± 0.8 μg/mL. The most active extract towards elastase release was from Hippocastani cortex which at concentration 10 μg/mL showed 62.0 ± 6.9% inhibition. CONCLUSION Anti-hyaluronidase and anti-elastase activity of chosen tannin-rich plant materials can support their traditional use in folk medicine. Strong inhibition of both enzymes by extract from Lythri herba makes this pharmacopeial plant material an interesting topic for further biological and phytochemical examination.

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.

Oleuropein and oleacein may restore biological functions of endothelial progenitor cells impaired by angiotensin II via activation of Nrf2/heme oxygenase-1 pathway

Endothelial progenitor cells (EPCs) are responsible for neovascularization of ischaemic tissue and may participate in re-endothelization of an injured arterial wall. There is evidence that angiotensin II, by an increase of gp91phox expression and induction of ROS generation, accelerates cell senescence and impairs functions of EPCs. Oleacein is a main phenolic compound from olive oil, whereas oleuropein is present in olive leaves. Both compounds possess antioxidative, hypotensive and anti-inflammatory properties and show beneficial activity on the cardiovascular system. In this study, we examined whether oleoeuropein and oleacein could protect EPCs against impairment of their functions due to angiotensin-induced cell senescence. CD31(+)/VEGFR-2(+) cells were isolated from young healthy volunteers blood samples and cultured on fibronectin-coated plates with angiotensin (1.0μM) in presence or absence of increasing concentrations (from 1.0 to 10.0 μM) of oleoeuropein or oleacein. As compared to angiotensin II-treated cells, EPCs exposed to oleacein or oleuropein prior to angiotensin II showed a significant increase of proliferation and telomerase activity, and a decrease in the percentage of senescent cells and intracellular ROS formation. Oleacein and oleuropein restored migration, adhesion and tube formation of EPCs diminished by angiotensin II in a concentration-dependent manner. This effect was related to NF-E2-related factor 2 (Nrf2) transcription factor activation and the increase of heme oxygenase-1 (HO-1) expression.

Screening of traditional European herbal medicines for acetylcholinesterase and butyrylcholinesterase inhibitory activity

Screening of traditional European herbal medicines for acetylcholinesterase and butyrylcholinesterase inhibitory activity Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimers disease (AD) to enhance central cholinergic transmission. On the other hand, butyrylcholinesterase (BuChE) inhibitors were reported to produce a significant increase in brain extracellular AChE without triggering severe peripheral or central side effects. In the present study, we selected twelve plants used in traditional European medicine to treat different central nervous system (CNS) disorders or to improve memory. Methanolic and hexane extracts of these plants were tested for the AChE and BuChE inhibitory activity using Ellmans colorimetric method. The most potent AChE and BuChE inhibition was observed in the hexane extracts of the flowers of Arnica chamissonis Less. subs. foliosa and Ruta graveolens L. herb at a concentration of 400 μg mL-1. However, methanolic extracts of the flowers of Arnica chamissonis Less. subs. foliosa and the Hypericum perforatum L. herb demonstrated at the same concentration, selective inhibition only against AChE but not against BuChE. The other extracts did not show any significant AChE or BuChE inhibitory activity. Our results show that further investigations of the extracts of arnica, rue and St. Johns Wort are needed to identity the compounds responsible for the AChE and BuChE inhibitory activity. Pretraživanje tradicionalnih europskih ljekovitih biljaka na inhibiciju acetilkolinesteraze i butirilkolinesteraze Inhibitori acetilkolinesteraze (AChE) povećavaju kolinergičku transmisiju u mozgu, pa se koriste za simptomatsko liječenje Alzheimerove bolesti (AD). S druge strane, inhibitori butirilkolinesteraze (BuChE) značajno povećavaju ekstracelularnu količinu AChE u mozgu, a da pri tome ne uzrokuju snažne nuspojave ni u središnjem ni i perifernom živčanom sustavu. Galantamin, jedan od odobrenih AChE inhibitora, alkaloid iz lukovica narcisa, pokazuje da su biljke značajni izvor novih potencijalnih AChE- i BuChE-inhibitora. U ovom radu, ispitivan je učinak dvanaest biljaka koje se koriste u tradicionalnoj europskoj medicini na različite poremećaje središnjeg živčanog sustava i na poboljšanje pamćenja. Pomoću Ellmanove kolorimetrijske metode praćen je inhibitorni učinak metanolnih i heksanskih ekstrakata tih biljaka na AChE i BuChE. Najjači inhibitorni učinak pokazali su heksanski ekstrakti cvjetova Arnica chamissonis Less. susb. foliosa i nadzemnih dijelova Ruta graveolens L. u koncentraciji od 400 μg mL-1. Medutim, metanolni ekstrakti cvjetova Arnica chamissonis Less. susb. foliosa i nadzemnih dijelova Hypericum perforatum L. u istim koncentracijama pokazuju selektivnu inhibiciju samo na AChE. Ostali ekstrakti bili su nedjelotvorni. Rezultati ukazuju na potrebu daljnjih ispitivanja ekstrakata arnike, rute i gospine trave da se utvrdi koji su sastojci ekstrakata odgovorni za inhibiciju AChE i BuChE.

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.

Novel Biological Properties of Oenothera paradoxa Defatted Seed Extracts: Effects on Metallopeptidase Activity

In this study, for the first time, we used the in vitro metallopeptidase model for the identification of a potential novel activity of defatted evening primrose seed extracts. Prepared extracts of different polarity (aqueous, 60% ethanolic, isopropanolic, and 30% isopropanolic) at concentrations of 1.5-100 microg/mL exhibited a significant and dose dependent inhibition of three tested enzymes. The 50% inhibition of enzymes activity showed that aminopeptidase N (APN) was the enzyme affected to the greatest extent with IC50 at the level of 2.8 microg/mL and 2.9 microg/mL for aqueous and 30% isopropanolic extracts, respectively. The activity of neutral endopeptidase (NEP) was quite strongly inhibited by the extracts as well. The HPLC-DAD analysis and bioguided fractionation led to the identification of four active compounds: (-)-epicatechin gallate, proanthocyanidin B3, oenothein B, and penta-O-galloyl-beta-D-glucose (PGG). Oenothein B has been shown previously to inhibit metallopeptidases. The three other compounds are known to inhibit angiotensin-converting enzyme (ACE), but they have not been previously reported to inhibit the NEP and APN activity. PGG and procyanidins with different degrees of polymerization, as the dominating compounds in O. paradoxa seeds, seemed to play a role in the crude extract activity.

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.

Dual inhibition of metallopeptidases ACE and NEP by extracts, and iridoids from Ligustrum vulgare L.

AIM OF THE STUDY In order to confirm the traditional use of Ligustrum vulgare L. (common privet, Oleaceae) we investigated the inhibitory activity of different extracts from leaves (LlE), flowers (LflE) and fruits (LfrE) on metallopeptidases ACE and NEP. MATERIALS AND METHODS Powdered plant materials were first extracted with water and then with ethyl acetate and n-butanol saturated with water. The metallopeptidases activity was determined using in vitro fluorimetric assays. RESULTS At a concentration of 100microg/ml the ethyl acetate extracts showed the highest activity. The bio-guided fractionation of the leaves extract led to the isolation of two iridoids which were identified by (1)H, (13)C and HETCOR NMR spectroscopy as oleuropein and ligstroside aglycones. Both compounds are dual ACE/NEP inhibitors with IC(50) of 20 and 25microM for ACE and IC(50) of 35 and 75microM for NEP, respectively. Secoirydoids glycosides, tyrozol and hydroxytyrozol, as well as, flavonoids present in the ethyl acetate extracts showed little or no inhibitory activity. CONCLUSIONS Our results partially support the diuretic and hypotensive activities of common privet.