Inhibition of SARS-CoV-2 infection and replication by Petasites hybridus CO2-extract (Ze 339).

BACKGROUND: The intensified search for low-threshold herbal anti-viral drugs would be of great advantage in prevention of early stages of infection. Since the SARS-CoV-2 Omicron variant has prevailed in western countries, the course has only been mild, but there are still no widely available drugs that can alleviate or shorten disease progression and counteract the development of Long-COVID. This study aimed to investigate the antiviral effects of a CO2-extract from Petasites hybridus (Ze 339). METHODS: We analyzed the infection and replication rate of SARS-CoV-2 in primary normal human bronchial epithelial cells (NHBEs) using a GFP-expressing version of the wild-type SARS-CoV-2 virus and live cell imaging. Upon infection with a clinical isolate of the Omicron variant, viral RNA content was quantified, and plaque assays were performed. In addition, the human transcriptome was analyzed after 4- and 24-hours post infection using whole genome microarrays. RESULTS: Ze 339 had a protective effect on primary airway epithelial cells during SARS-CoV-2 infection and impeded SARS-CoV-2 infection and replication in NHBE. Notably, Ze 339 inhibited the expression of infection-induced IFNA10 by 8.6-fold (p < 0.05) and additionally reduced a wide range of other interferons (IFNA6, IFNA7, IFNA8, IFNA21, IFNE, IFNW1). CONCLUSION: Thereby, Ze 339 attenuated epithelial infection by SARS-CoV-2 and modeled the IFN response. In conclusion, this study highlights Ze 339 as a potential treatment option for COVID-19 that limits infection-associated cell intrinsic immune responses.

Impact of St. John's wort extract Ze 117 on stress induced changes in the lipidome of PBMC.

BACKGROUND: Membrane lipids have an important function in the brain as they not only provide a physical barrier segregating the inner and outer cellular environments, but are also involved in cell signaling. It has been shown that the lipid composition effects membrane fluidity which affects lateral mobility and activity of membrane-bound receptors. METHODS: Since changes in cellular membrane properties are considered to play an important role in the development of depression, the effect of St. John's wort extract Ze 117 on plasma membrane fluidity in peripheral blood mononuclear cells (PBMC) was investigated using fluorescence anisotropy measurements. Changes in fatty acid residues in phospholipids after treatment of cortisol-stressed [1 µM] PBMCs with Ze 117 [10-50 µg/ml] were analyzed by mass spectrometry. RESULTS: Cortisol increased membrane fluidity significantly by 3%, co-treatment with Ze 117 [50 µg/ml] counteracted this by 4.6%. The increased membrane rigidity by Ze 117 in cortisol-stressed [1 µM] PBMC can be explained by a reduced average number of double bonds and shortened chain length of fatty acid residues in phospholipids, as shown by lipidomics experiments. CONCLUSION: The increase in membrane rigidity after Ze 117 treatment and therefore the ability to normalize membrane structure points to a new mechanism of antidepressant action of the extract.

Indigenous Yeasts from Rose Oil Distillation Wastewater and Their Capacity for Biotransformation of Phenolics.

The indigenous yeasts associated with the spontaneous fermentation of phenolic-rich rose oil distillation wastewater (RODW) generated after the industrial distillation of rose oil were studied. The ITS-rDNA sequence analysis of the samples collected from RODW fermented at semi-sterile conditions, a waste deposition lagoon and endophytic yeasts isolated from industrially cultivated Rosa damascena suggests that the spontaneous RODW fermentation is caused by yeasts from the genus Cyberlindnera found also as endophytes in the rose flowers. Phylogenetic analysis based on the nucleotide sequences of the translation elongation factor (TEF1α) and 18S- and 26S- rRNA genes further confirmed the taxonomic affiliation of the RODW yeast isolates with the genus Cyberlindnera. The RODW fermentation capacity of a selected set of indigenous yeast isolates was studied and compared with those of common yeast strains. The indigenous yeast isolates demonstrated a superior growth rate, resulting in a nearly double reduction in the phenolic content in the fermented RODW. The indigenous yeasts' fermentation changed the RODW phenolics' composition. The levels of some particular phenolic glycosides decreased through the depletion and fermentation of their sugar moiety. Hence, the relative abundance of the corresponding aglycons and other phenolic compounds increased. The capacity for the biotransformation of RODW phenolics by indigenous yeasts is discussed.

Effect of St. John's wort extract Ze 117 on the lateral mobility of ß1-adrenergic receptors in C6 cells.

Depression has been associated with altered signal transduction of serotonergic, dopaminergic and adrenergic neurotransmitter systems in the brain. Signaling relies on receptor-ligand interactions and subsequent regulatory processes, but also on lateral receptor mobility. The aim of this study was to investigate the effect of the St. John's wort extract Ze 117 on the lateral mobility of SNAP-tagged ß1-adrenergic receptors (ß1AR) in the plasma membrane of C6 cells under both, non-stimulating and isoprenaline-stimulating conditions. Single particle tracking (SPT) was used, whereby the registered trajectories were evaluated by variational Bayesian treatment of a hidden Markov model (vbSPT) and packing coefficient (Pc) analysis with respect to diffusion coefficients, receptor state occupancies and confinement. Three different diffusion states were identified, differing in their diffusion coefficients. Treatment with Ze 117 [25 µg/ml] decreased the mobility of the ß1AR, which was manifested by a relative increase in the slow-diffusing state S1 (0.21-0.30) compared to control and by an increase in receptor confinement (79.4-68.1 nm). After isoprenaline stimulation of control cells, the slow-diffusing state was more pronounced, whereas confinement was not affected. In summary, SPT has been shown to be a powerful method to analyze lateral receptor mobility. Furthermore, the present study identified a correlation between Ze 117 treatment and ß1AR mobility.

Impact of the clinically approved Petasites hybridus extract Ze 339 on intestinal mechanisms involved in the handling of histamine.

In patients with histamine intolerance accumulated or ingested histamine causes a broad range of undesirable symptoms. Food-derived histamine is degraded by intestinal diamine oxidase (DAO) and histamine-N-methyltransferase (HNMT), while the organic cation transporter 3 (OCT3) contributes to the transcellular flux of histamine. Anecdotal evidence from patients with HIT suggests an improvement of symptoms related to histamine intolerance after intake of Ze 339, a lipophilic CO2-extract prepared from the leaves of Petasites hybridus. Thus, it was the aim of this study to investigate the influence of Ze 339 on DAO, HNMT and OCT3 using Caco-2 and MDCKII cells. Even though Ze 339 reduced mRNA levels of HNMT and DAO, there was no change in protein expression. Ze 339 changed neither the basal release nor the enzymatic activity of DAO. Testing the interaction of Ze 339 with the transcellular histamine transport, we observed a significant increase in the basal to apical flux in presence of high Ze 339 concentrations at the early phases of the experiment. Testing the influence of Ze 339 on OCT3-mediated histamine uptake in overexpressing MDCKII cells revealed a dose-dependent inhibition with an estimated IC50 of 26.9 ug/mL for the extract. In conclusion, we report an effect of Ze 339 on transcellular histamine transport, where inhibition of OCT3 may contribute.

The Petasites hybridus CO2 Extract (Ze 339) Blocks SARS-CoV-2 Replication In Vitro.

The coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (SARS-CoV-2), has spread worldwide, affecting over 250 million people and resulting in over five million deaths. Antivirals that are effective are still limited. The antiviral activities of the Petasites hybdridus CO2 extract Ze 339 were previously reported. Thus, to assess the anti-SARS-CoV-2 activity of Ze 339 as well as isopetasin and neopetasin as major active compounds, a CPE and plaque reduction assay in Vero E6 cells was used for viral output. Antiviral effects were tested using the original virus (Wuhan) and the Delta variant of SARS-CoV-2. The antiviral drug remdesivir was used as control. Pre-treatment with Ze 339 in SARS-CoV-2-infected Vero E6 cells with either virus variant significantly inhibited virus replication with IC50 values of 0.10 and 0.40 µg/mL, respectively. The IC50 values obtained for isopetasin ranged between 0.37 and 0.88 µM for both virus variants, and that of remdesivir ranged between 1.53 and 2.37 µM. In conclusion, Ze 339 as well as the petasins potently inhibited SARS-CoV-2 replication in vitro of the Wuhan and Delta variants. Since time is of essence in finding effective treatments, clinical studies will have to demonstrate if Ze339 can become a therapeutic option to treat SARS-CoV-2 infections.

Cimicifuga racemosa Extract Ze 450 Re-Balances Energy Metabolism and Promotes Longevity.

Recently, we reported that the Cimicifuga racemosa extract Ze 450 mediated protection from oxidative cell damage through a metabolic shift from oxidative phosphorylation to glycolysis. Here, we investigated the molecular mechanisms underlying the effects of Ze 450 against ferroptosis in neuronal cells, with a particular focus on mitochondria. The effects of Ze 450 on respiratory complex activity and hallmarks of ferroptosis were studied in isolated mitochondria and in cultured neuronal cells, respectively. In addition, Caenorhabditis elegans served as a model organism to study mitochondrial damage and longevity in vivo. We found that Ze 450 directly inhibited complex I activity in mitochondria and enhanced the metabolic shift towards glycolysis via cMyc and HIF1α regulation. The protective effects against ferroptosis were mediated independently of estrogen receptor activation and were distinct from effects exerted by metformin. In vivo, Ze 450 protected C. elegans from the mitochondrial toxin paraquat and promoted longevity in a dose-dependent manner. In conclusion, Ze 450 mediated a metabolic shift to glycolysis via direct effects on mitochondria and altered cell signaling, thereby promoting sustained cellular resilience to oxidative stress in vitro and in vivo.

Extract matrix composition does not affect in vitro leukotriene inhibitory effects of the Petasites hybridus extract Ze 339.

It has been shown that a lipophilic CO2-extract prepared from the leaves of Petasites hybridus (Ze 339) inhibited leukotriene synthesis in vitro and ex vivo. The inhibition of the leukotriene synthesis was solely attributed to the sum of the petasins, namely petasin and its isomers isopetasin and neopetasin. To further investigate the influence of the extract matrix on leukotriene synthesis inhibition, we compared twelve selected batches of Ze 339 that differed significantly in the composition of the extract matrix. Quantitative analysis of the twelve extract batches revealed high contents of petasins [28.8-41.9%], fatty acids [17.1-27.2%] and crude oil and fat [17.7-44.2%]. The amount of sterols ranged between 3.0 and 4.9% and that of essential oils between 1.3 and 10.5%. Based on the quantitative analysis, 97-100% of the extract mass could be attributed to the above mentioned groups of ingredients. Despite significant differences in extract matrix composition, only the content of petasins was critical for the dose-dependent inhibition of leukotriene synthesis. However, at equal concentrations of petasins, no significant differences in 5-LOX, LTC4 synthase and LTA4 hydrolase inhibition were detected between the selected extract batches, despite differences in the composition of the petasin isomers. Our data suggest that the extract matrix of Ze 339 has no effect on leukotriene inhibitory effects of the petasins.

Treatment of Early Allergic and Late Inflammatory Symptoms of Allergic Rhinitis with Petasites hybridus Leaf Extract (Ze 339): Results of a Noninterventional Observational Study in Switzerland.

The primary objective of this noninterventional, observational study was to assess the effectiveness of the Petasites hybridus leaf extract (Ze 339) on early allergic and late inflammatory symptoms of allergic rhinitis in Swiss outpatients. This study was conducted by general practitioners and allergologists. Data from 226 patients were collected during three documented visits. The intermediate visit was ideally made 2-4 weeks after the baseline visit, followed by the final visit approximately 2-4 months later. The mean study duration was 63 days, with 75% of patients being treated for at least 4 weeks. Of the patients, 58.5% started with Ze 339 monotherapy, and 41.5% received other antiallergic and/or sympathomimetic drugs. In both groups, the allergic total symptom score and the inflammatory total symptom scores were significantly (p < 0.001) reduced, and the scores for quality of life were improved. Both physicians and patients were very satisfied with the treatment and the concept of therapy, not only for short-term (seasonal) therapy but also for long-term therapy. The tolerability was good: only three mild gastrointestinal adverse events occurred. In summary, the effectiveness of P. hybridus leaf extract Ze 339 for the treatment of early allergic and late inflammatory symptoms of allergic rhinitis could be confirmed.

Saponins from Saffron Corms Inhibit the Gene Expression and Secretion of Pro-Inflammatory Cytokines.

Corms are obtained as a byproduct during the cultivation of saffron (Crocus sativus). In a project aimed at the valorization of this waste product, we observed that a 70% EtOH extract of the corms and a sugar-depleted MeOH fraction of the extract inhibited the TNF-α/IFN-γ-induced secretion and gene expression of the chemokines IL-8, MCP-1, and RANTES in human HaCaT cells. The effects were in part stronger than those of the positive control hydrocortisone. For preparative isolation, the 70% EtOH extract was partitioned between n-BuOH and water. Separation of the n-BuOH-soluble fraction by centrifugal partition chromatography, followed by preparative and semipreparative HPLC, afforded a series of bidesmosidic glycosides of echinocystic acid bearing a 3,16-dihydroxy-10-oxo-hexadecanoic acid residue attached to the glycosidic moiety at C-28. They include azafrines 1 and 2, previously reported in saffron, and eight new congeners named azafrines 3-10. Saffron saponins significantly inhibited TNF-α/IFN-γ-induced secretion of RANTES in human HaCaT cells at 1 µM (p < 0.001). Some of them further lowered TNF-α/IFN-γ-induced gene expression.

Antiproliferative Effects of Pterodon pubescens Extract and Isolated Diterpenes in HaCaT Cells.

Pterodon pubescens fruits are popularly used because of their analgesic and anti-inflammatory actions, which are attributed to the isolated compounds with a vouacapan skeleton. This work aimed to evaluate the antiproliferative and anti-inflammatory effects of a P. pubescens fruit dichloromethane extract and the vouacapan diterpene furan isomer's mixture (1 : 1) (6α-hydroxy-7ß-acetoxy-vouacapan-17ß-oate methyl ester and 6α-acetoxy-7ß-hydroxy-vouacapan-17ß-oate methyl ester isomers) in HaCaT cells using the cell migration and the BrDU incorporation assay. Levels of IL-8 were measured by ELISA after TNF-α stimulation. HPLC/DAD analysis of the extract revealed the expressive presence of vouacapan diterpene furan isomer's mixture. P. pubescens extract (1.5625 - 25 µg/mL) and vouacapan diterpene furan isomer's mixture (3.125 - 50 µM) inhibited cell proliferation as indicated by a decreased BrdU-incorporation. For the evaluation of cell migration, time-lapse microscopy was used. P. pubescens presented inhibition on cell migration at all concentrations tested (3.125 - 12.5 µg/mL), whereas for the VDFI mixture, the inhibition was only observed at the highest concentrations (12.5 and 25 µM) tested. Furthermore P. pubescens extract and vouacapan diterpene furan isomer's mixture significantly decreased IL-8 levels. Our results showed antiproliferative and anti-inflammatory effects on HaCaT cells treated with the extract and the vouacapan isomer's mixture, without affecting cell viability. These activities could be attributed to the voucapan molecular structures. In conclusion, topical products developed of P. pubescens extract or the voucapan isomer's mixture should be further studied as a potential product for local treatment against hyperproliferative lesions as in psoriasis vulgaris, representing an alternative treatment approach.

Honeybush Extracts (Cyclopia spp.) Rescue Mitochondrial Functions and Bioenergetics against Oxidative Injury.

Mitochondrial dysfunction plays a major role not only in the pathogenesis of many oxidative stress or age-related diseases such as neurodegenerative as well as mental disorders but also in normal aging. There is evidence that oxidative stress and mitochondrial dysfunction are the most upstream and common events in the pathomechanisms of neurodegeneration. Cyclopia species are endemic South African plants and some have a long tradition of use as herbal tea, known as honeybush tea. Extracts of the tea are gaining more scientific attention due to their phenolic composition. In the present study, we tested not only the in vitro mitochondria-enhancing properties of honeybush extracts under physiological conditions but also their ameliorative properties under oxidative stress situations. Hot water and ethanolic extracts of C. subternata, C. genistoides, and C. longifolia were investigated. Pretreatment of human neuroblastoma SH-SY5Y cells with honeybush extracts, at a concentration range of 0.1-1 ng/ml, had a beneficial effect on bioenergetics as it increased ATP production, respiration, and mitochondrial membrane potential (MMP) after 24 hours under physiological conditions. The aqueous extracts of C. subternata and C. genistoides, in particular, showed a protective effect by rescuing the bioenergetic and mitochondrial deficits under oxidative stress conditions (400 µM H2O2 for 3 hours). These findings indicate that honeybush extracts could constitute candidates for the prevention of oxidative stress with an impact on aging processes and age-related neurodegenerative disorders potentially leading to the development of a condition-specific nutraceutical.

Single dose pharmacokinetics of intravenous 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid in rats.

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (3-HPAA) are intestinal metabolites of the dietary flavonoid quercetin. DOPAC reportedly showed anxiolytic activity after i.p. administration in rats. The fate of these metabolites after consumption, and the pharmacological properties of 3-HPAA in the body are largely unknown. The aim of the current study was to characterize pharmacokinetic properties of DOPAC and 3-HPAA after intravenous bolus application in rats. UHPLC-MS/MS methods for quantification of DOPAC and 3-HPAA levels in lithium heparin Sprague Dawley rat plasma were developed and validated according to international regulatory guidelines. Non-compartmental and compartmental analyses were performed. Pharmacokinetic profiles of DOPAC and 3-HPAA followed a two-compartment body model, with a fast distribution into peripheral tissues (half-lives of 3.27-5.26 min) and rapid elimination from the body (half-lives of 18.4-33.3 min).

In vitro hepatotoxicity of Petasites hybridus extract (Ze 339) depends on the concentration, the cytochrome activity of the cell system, and the species used.

Ze 339, a CO2 extract prepared from the leaves of Petasites hybridus, possesses antispasmodic and anti-inflammatory effects and is proven to be effective in the treatment of allergic rhinitis. To study possible hepatotoxic effects of Ze 339, its main constituents and metabolites, a series of in vitro investigations were performed. Furthermore, different reconstituted fractions of extract (petasins and fatty acid fraction) were examined in three in vitro test systems using hepatocytes: Two human cell lines, with lower and higher activity of cytochrome P450 enzymes (HepG2, HepaRG) as well as a rodent cell line with high cytochrome P450 activity (H-4-II-E), were used. Metabolic activity, assessed by the WST-1 assay, was chosen as indicator of cytotoxicity. To assess potential bioactivation of Ze 339 compounds, metabolic experiments using S9 fractions from rats, dogs, and humans and isolated cytochromes (human/rat) were performed, and the formation of reactive metabolites was assessed by measuring cellular concentrations of glutathione and glutathione disulphide. Our data revealed that the cytotoxicity of Ze 339, its single constituents, and main metabolites depends on the concentration, the cytochrome activity of the cell system, and the species used.

Clinical relevance of St. John's wort drug interactions revisited.

The first clinically relevant reports of preparations of St. John's wort (SJW), a herbal medicine with anti-depressant effects, interacting with other drugs, altering their bioavailability and efficacy, were published about 20 years ago. In 2000, a pharmacokinetic interaction between SJW and cyclosporine caused acute rejection in two heart transplant patients. Since then, subsequent research has shown that SJW altered the pharmacokinetics of drugs such as digoxin, tacrolimus, indinavir, warfarin, alprazolam, simvastatin, or oral contraceptives. These interactions were caused by pregnane-X-receptor (PXR) activation. Preparations of SJW are potent activators of PXR and hence inducers of cytochrome P450 enzymes (most importantly CYP3A4) and P-glycoprotein. The degree of CYP3A4 induction correlates significantly with the hyperforin content in the preparation. Twenty years after the first occurrence of clinically relevant pharmacokinetic drug interactions with SJW, this review revisits the current knowledge of the mechanisms of action and on how pharmacokinetic drug interactions with SJW could be avoided. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.

A FLIPR Assay for Discovery of GABAA Receptor Modulators of Natural Origin.

A fluorometric imaging plate reader (FLIPR) assay utilizing Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α 1 ß 2 γ 2 subunit composition was evaluated and validated for rapid screening of plant extract libraries and efficient localization of active compounds in extracts. Validation was performed with pure compounds and extracts known to contain allosteric GABAA receptor modulators. Plants extracts that had been previously reported as active in an assay using Xenopus laevis oocytes transiently expressing GABAA receptors of α 1 ß 2 γ 2 subunit composition were also active in the FLIPR assay. A protocol for HPLC-based activity profiling was developed, whereby separations of 0.4 - 1.2 mg of extracts on an analytical HPLC column were found to be sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of known GABAergic natural products, such as magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extract. EC50 values of compounds (magnolol: 4.81 ± 1.0 µM, valerenic acid: 12.56 ± 1.2 µM, and piperine: 5.76 ± 0.7 µM) were found to be comparable or lower than those reported using Xenopus oocyte assays.

Permeation characteristics of hypericin across Caco-2 monolayers in the presence of single flavonoids, defined flavonoid mixtures or Hypericum extract matrix.

OBJECTIVES: The major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the Caco-2 cell system when combined with various flavonoids, mixtures of flavonoids or Hypericum perforatum extract matrix (STW3-VI). METHODS: The permeation characteristics of hypericin in the absence or presence of quercetin, quercitrin, isoquercitrin, hyperoside and rutin were tested. Hypericin (5 µm) was mixed with single flavonoids (20 µm) or with different flavonoid combinations (each flavonoid 4 or 10 µm, total flavonoid concentration: 20 µm). Further, the uptake of hypericin (5 µm) in the presence of H. perforatum extract matrix (7.25, 29 and 58 µg/ml) was studied. KEY FINDINGS: Following application of hypericin to the apical side of the monolayer, only negligible amounts of the compound were found in the basolateral compartment. From all tested flavonoids, only quercitrin increased the basolateral amount of hypericin. Dual flavonoid combinations were not superior compared to the single combinations. The amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of extract matrix (from 0 to 7.5%). CONCLUSION: Comparing the effects of various flavonoid mixtures vs the extract matrix, it can be concluded that, besides flavonoids, the extract seems to contain further compounds (e.g. phenolic acids or proanthocyanidins) which substantially improve the permeation characteristics of hypericin.

Hyperforin and Miquelianin from St. John's Wort Attenuate Gene Expression in Neuronal Cells After Dexamethasone-Induced Stress.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis plays an important part in the development of depressive symptoms. In this study, the effects of a commercial St. John's wort extract (STW3-VI), hyperforin, miquelianin, and the selective serotonin reuptake inhibitor citalopram on the expression of genes relevant to HPA axis function were investigated in human neuronal cells. SH-SY5Y cells were treated with STW3-VI (20 µg/mL), hyperforin (1 µM), miquelianin (10 µM), or citalopram (10 µM) in the presence of the glucocorticoid receptor agonist dexamethasone (DEX,10 µM) for 6 h and 48 h, respectively. Quantitative real-time polymerase chain reaction was used to determine the expression of FKBP5 (FK506 binding protein 51), CREB (cAMP responsive element binding protein), GRIK4 (glutamate ionotropic receptor kainate type subunit 4), VEGF (vascular endothelial growth factor), NET (norepinephrine transporter), and ARRB (ß-arrestins), promising biomarkers of antidepressant therapy. Using DEX to mimic stress conditions, it was shown that the gene expression pattern of FKBP5, CREB, GRIK4, VEGF, NET, and ARRB2 in SH-SY5Y cells is time- and treatment-dependent. Most pronounced effects were observed for FKBP5: after 6 h of co-incubation, only STW3-VI could reverse the DEX-induced increase in FKBP5 expression, and after 48 h, citalopram, miquelianin, and hyperforin also reversed the glucocorticoid-induced increase in FKBP5 mRNA expression. The effects observed on FKBP5, CREB, GRIK4, VEGF, NET, and ARRB2 are in good correlation with published data, suggesting that this in vitro model could be used to screen the responsiveness of antidepressants under stress conditions.

Metabolite Profile and Antiproliferative Effects in HaCaT Cells of a Salix reticulata Extract.

Phenolic constituents of Salix reticulata (Salicaceae) and antiproliferative activity of an extract and individual compounds were investigated in immortalized human non-tumorigenic keratinocytes (HaCaT). A MeOH extract from aerial parts afforded several flavonoids, including luteolin and apigenin glycosides (2-5 and 9) and catechin (1), two procyanidin fractions, and the phenolic glucosides picein (6), triandrin (7), and salicortin (8). In an adenosine triphosphate assay, the MeOH extract reduced cell viability by approximately 60 % at a concentration of 100 µg/mL. Cell proliferation was assessed with a BrdU incorporation ELISA assay. The extract inhibited proliferation of HaCaT cells in a concentration-dependent manner, with approximately 50 % inhibition at 100 µg/mL. In time-lapse assays, the extract showed distinct inhibitory effects on cell migration at concentrations of 12.5, 25, and 50 µg/mL. The activity of selected constituents was also determined. Luteolin-7-O-ß-glucuronide (3) significantly inhibited cell proliferation at concentrations of 10 and 50 µM. In contrast, luteolin-7-O-ß-glucopyranoside (2) and a procyanidin fraction (P1) had only weak effects, while picein (6) and salicortin (8) did not affect cell proliferation. Luteolin-7-O-ß-glucuronide (10 µM) and, to a lesser extent, the procyanidin fraction (10 µg/mL) also inhibited cell migration.