Evaluation of the Light-Sensitive Cytotoxicity of Hypericum perforatum Extracts, Fractions, and Pure Compounds

Hypericum perforatum (Hp) is known for possessing antidepressant and antiviral activities. Despite its use as an alternative to conventional antidepressants, the identification of the cytotoxic chemicals derived from this herb is incomplete. In this study, the cytotoxicity of Hp extracts prepared in solvents ranging in polarity, fractions of one extract, and purified compounds were examined in three cell lines. All extracts exhibited significant cytotoxicity; those prepared in ethanol (no hyperforin, 3.6 μM hypericin, and 134.6 μM flavonoids) showed between 7.7 and 77.4% cell survival (p \u3c 0.0001 and 0.01), whereas the chloroform and hexane extracts (hyperforin, hypericin, and flavonoids not detected) showed approximately 9.0 (p \u3c 0.0001) and 4.0% (p \u3c 0.0001) survival. Light-sensitive toxicity was observed primarily with the ethanol extracts sequentially extracted following removal of material extracted in either chloroform or hexane. The absence of light-sensitive toxicity with the Hp extracts suggests that the hypericins were not playing a prominent role in the toxicity of the extracts

Characterizing the Metabolic Fingerprint and Anti-inflammatory Activity of Hypericum gentianoides

In this paper we characterize the metabolic fingerprint and first reported anti-inflammatory activity of Hypericum gentianoides. H. gentianoides has a history of medical use by Native Americans, but it has been studied very little for biological activity. High-performance liquid chromatography (HPLC) and liquid chromatography−electrospray ionization−mass spectrometry (LC-ESI-MS) analyses of a methanol extract show that H. gentianoides contains a family of over nine related compounds that have retention times, mass spectra, and a distinctive UV absorption spectra characteristic of certain acyl-phloroglucinols. These metabolites are abundant relative to other secondary products present in H. gentianoides, accounting for approximately 0.2 g per gram of dry plant tissue. H. gentianoides methanol extracts and a specific semipreparative HPLC fraction from these extracts containing the putative acyl-phloroglucinols reduce prostaglandin E2 synthesis in mammalian macrophages

Transgenic Alfalfa That Accumulates Piceid (Trans-Resveratrol-3-O−β-D-glucopyranoside) Requires the Presence of β -Glucosidase to Inhibit the Formation of Aberrant Crypt Foci in the Colon of CF-1 Mice

Plants have been genetically enhanced to produce a number of products for agricultural, industrial and pharmaceutical purposes. This technology could potentially be applied to providing chemoprevention strategies to the general population. Resveratrol (3,5,4′-trihydroxystilbene) is a compound that has been shown to have protective activity against a number of cancers and could be an ideal candidate for such an application. Alfalfa that was genetically modified to express resveratrol-synthase was used as a model in applying biotechnological approaches to cancer prevention. The transgenic alfalfa, which accumulates resveratrol as a glucoside (piceid = trans-resveratrol-3-O−β-D-glucopyranoside) (152 ± 17.5 μ g piceid/g dry weight), was incorporated into a standard mouse diet at 20% of the diet by weight and fed for 5 wk to 6-wk-old, female CF-1 mice (N = 17–30) that were injected with a single dose of azoxymethane (5 mg/kg body weight). While the addition of resveratrol-aglycone (20 mg/kg diet) to the basal diet reduced the number of aberrant crypt foci/mouse, the transgenic alfalfa did not inhibit the number, size, or multiplicity of aberrant crypt foci in the colon of the CF-1 mice relative to control alfalfa which does not accumulate resveratrol-glucoside. However, diets containing transgenic alfalfa with an exogenous β -glucosidase (860 U/kg diet) did significantly inhibit the number of aberrant crypt foci in the distal 2 cm of the colon of the mice relative to mice fed diets containing the transgenic alfalfa without the enzyme (P \u3c 0.05; Fisher\u27s Combination of p-values). The β -glucosidase alone appeared to have no effect on the inhibition of aberrant crypt foci. These results suggest that piceid in transgenic piceid-accumulating alfalfa was not bioavailable

Dietary energy restriction, in part through glucocorticoid hormones, mediates the impact of 12-O-tetradecanoylphorbol-13-acetate on jun D and fra-1 in sencar mouse epidermis

Dietary energy restriction (DER, 40% calorie reduction from fat and carbohydrate) inhibited mouse skin carcinogenesis and decreased 12-O-tetradecanoyl-13-phorbol acetate (TPA)-induced activator protein-1 (AP-1):DNA binding previously. This study measured protein levels of c-jun, jun B, jun D, c-fos, fra-1, and fra-2 and examined their contribution to AP-1:DNA binding by electrophoretic mobility shift assay (EMSA) with supershift analysis in the epidermis of control and DER Sencar mice exposed to TPA. TPA significantly increased c-jun, jun B, c-fos, fra-1, and fra-2 and decreased jun D within 3–6 h after treatment. AP-1:DNA binding reached a maximum 2.5-fold induction over controls 4 h after TPA treatment and antibodies to jun B, jun D, and fra-2 in the EMSA binding reaction resulted in supershifts in both acetone- and TPA-treated mice 1–6 h after treatment. The effect of corticosterone (CCS) and DER on the AP-1 proteins and on the composition of the AP-1:DNA complex was measured in adrenalectomized (adx) mice. DER reduced the TPA impact on jun D and enhanced the induction of fra-1. In addition, CCS-supplemented groups had significantly lower jun D and higher fra-2 than adx groups and sham groups. While sham animals treated with either acetone or TPA contained jun B, jun D, and fra-2 proteins in the AP-1:DNA complex by supershift analysis, fra-2 was no longer seen in adx DER animals. In summary, our study supports potential roles for jun D, jun B, and fra-1 in the DER regulation of AP-1 function in the Sencar mouse skin carcinogenesis model

Inhibition of Prostaglandin E2 Production by Anti-inflammatory Hypericum perforatum Extracts and Constituents in RAW264.7 Mouse Macrophage Cells

Hypericum perforatum (Hp) is commonly known for its antiviral, antidepressant, and cytotoxic properties, but traditionally Hp was also used to treat inflammation. In this study, the anti-inflammatory activity and cytotoxicity of different Hp extractions and accessions and constituents present within Hp extracts were characterized. In contrast to the antiviral activity of Hp, the anti-inflammatory activity observed with all Hp extracts was light-independent. When pure constituents were tested, the flavonoids, amentoflavone, hyperforin, and light-activated pseudohypericin, displayed anti-inflammatory activity, albeit at concentrations generally higher than the amount present in the Hp extracts. Constituents that were present in the Hp extracts at concentrations that inhibited the production of prostaglandin E2 (PGE2) were pseudohypericin and hyperforin, suggesting that they are the primary anti-inflammatory constituents along with the flavonoids, and perhaps the interactions of these constituents and other unidentified compounds are important for the anti-inflammatory activity of the Hp extracts

Bauer ketones 23 and 24 from Echinacea paradoxa var. paradoxa inhibit lipopolysaccharide-induced nitric oxide, prostaglandin E2 and cytokines in RAW264.7 mouse macrophages

Among the nine Echinacea species, E. purpurea, E. angustifolia and E. pallida, have been widely used to treat the common cold, flu and other infections. In this study, ethanol extracts of these three Echinaceaspecies and E. paradoxa, including its typical variety, E. paradoxa var. paradoxa, were screened in lipopolysaccharide (LPS)-stimulated macrophage cells to assess potential anti-inflammatory activity. E. paradoxa var. paradoxa, rich in polyenes/polyacetylenes, was an especially efficient inhibitor of LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by 46%, 32%, 53% and 26%, respectively, when tested at 20 μg/ml in comparison to DMSO control. By bioactivity-guided fractionation, pentadeca-8Z-ene-11, 13-diyn-2-one (Bauer ketone 23) and pentadeca-8Z, 13Z-dien-11-yn-2-one (Bauer ketone 24) from E. paradoxa var. paradoxa were found primarily responsible for inhibitory effects on NO and PGE2 production. Moreover, Bauer ketone 24 was the major contributor to inhibition of inflammatory cytokine production in LPS-induced mouse macrophage cells. These results provide a rationale for exploring the medicinal effects of the Bauer ketone-rich taxon, E. paradoxa var.paradoxa, and confirm the anti-inflammatory properties of Bauer ketones 23 and 24

Pseudohypericin is necessary for the Light-Activated Inhibition of Prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E2 production (PGE2)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1 μM chlorogenic acid, 0.08 μM amentoflavone, 0.07 μM quercetin, and 0.03 μM pseudohypericin) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE2 of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A2, two enzymes in the PGE2-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), and the Hp fraction inhibited the antiinflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.

Endogenous Levels of Echinacea Alkylamides and Ketones Are Important Contributors to the Inhibition of Prostaglandin E2 and Nitric Oxide Production in Cultured Macrophages

Because of the popularity of Echinacea as a dietary supplement, researchers have been actively investigating which Echinacea constituent or groups of constituents are necessary for immunemodulating bioactivities. Our prior studies indicate that alkylamides may play an important role in the inhibition of prostaglandin E2 (PGE2) production. High-performance liquid chromatography fractionation, employed to elucidate interacting anti-inflammatory constituents from ethanol extracts of Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, and Echinacea tennesseensis, identified fractions containing alkylamides and ketones as key anti-inflammatory contributors using lipopolysaccharideinduced PGE2 production in RAW264.7 mouse macrophage cells. Nitric oxide (NO) production and parallel cytotoxicity screens were also employed to substantiate an anti-inflammatory response. E. pallida showed significant inhibition of PGE2 with a first round fraction, containing gas chromatography-mass spectrometry (GC-MS) peaks for Bauer ketones 20, 21, 22, 23, and 24, with 23 and 24 identified as significant contributors to this PGE2 inhibition. Chemically synthesized Bauer ketones 21 and 23 at 1 μM each significantly inhibited both PGE2 and NO production. Three rounds of fractionation were produced from an E. angustifolia extract. GC-MS analysis identified the presence of Bauer ketone 23 in third round fraction 3D32 and Bauer alkylamide 11 making up 96% of third round fraction 3E40. Synthetic Bauer ketone 23 inhibited PGE2 production to 83% of control, and synthetic Bauer alkylamide 11 significantly inhibited PGE2 and NO production at the endogenous concentrations determined to be present in their respective fraction; thus, each constituent partially explained the in vitro anti-inflammatory activity of their respective fraction. From this study, two key contributors to the anti-inflammatory properties of E. angustifolia were identified as Bauer alkylamide 11 and Bauer ketone 23

Hypericum in infection: Identification of anti-viral and anti-inflammatory constituents

The Iowa Center for Research on Botanical Dietary Supplements seeks to optimize Echinacea, Hypericum, and Prunella botanical supplements for human-health benefit, emphasizing anti-viral, anti-inflammatory, and anti-pain activities. This mini-review reports on ongoing studies on Hypericum. The Center uses the genetically diverse, well-documented Hypericum populations collected and maintained at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS), and the strength of research in synthetic chemistry at Iowa State University to tap natural diversity, to help discover key constituents and interactions among constituents that impact bioactivity and toxicity. The NCRPIS has acquired more than 180 distinct populations of Hypericum, with a focus on Hypericum perforatum L. (Hypericaceae), representing about 13% of currently recognized taxa. Center chemists have developed novel synthetic pathways for key flavones, acyl phloroglucinols, hyperolactones, and a tetralin that have been found in Hypericum, and these compounds are used as standards and for bioactivity studies. Both light-dependent and light-independent anti-viral activities have been identified by using bioactivity-guided fractionation of H. perforatum and a HIV-1 infection test system. Our Center has focused on light-independent activity, potentially due to novel chemicals, and polar fractions are undergoing further fractionation. Anti-inflammatory activity has been found to be light-independent, and fractionation of a flavonoid-rich extract revealed four compounds (amentoflavone, chlorogenic acid, pseudohypericin, and quercetin) that interacted in the light to inhibit lipopolysaccharide-induced prostaglandin E2 activity. The Center continues to explore novel populations of H. perforatum and related species to identify constituents and interactions of constituents that contribute to potential health benefits related to infection

Care-home Nurses' responses to the COVID-19 pandemic: Managing ethical conundrums at personal cost: A qualitative study

Introduction: The COVID-19 pandemic had an unprecedented effect on those living and working in care-homes for older people, as residents were particularly vulnerable to contracting the SARS-CoV-2 virus, associated with high morbidity and mortality. Often undervalued, care-home nurses (RNs) are leaders, managing complex care while working in isolation from their professional peers. The pandemic made this more apparent, when care and treatments for COVID-19 were initially unknown, isolation increased due to withdrawal of many professional health services, accompanied by staff shortages. Objective: To explore RNs' experiences of working in older people's care-homes during the COVID-19 pandemic. Design: Qualitative interview study. Setting: Care-homes for older people in England and Scotland, UK. Methods: Recruitment via direct contact with care-homes, social media, and links provided by national partners, then purposive sampling for age, gender, type of care-home, and location. Data collected through one-to-one online interviews using topic guide developed collaboratively with care-home nurses, focusing on how COVID-19 impacted on nurses' resilience and mental wellbeing. Data analyzed thematically using Tronto's ethics of care framework to guide development of interpretative themes. Results: Eighteen nurses (16 female; 16 adult, and two mental health nurses) were interviewed March–June 2021; majority aged 46–55 years; mean time registered with Nursing and Midwifery Council: 19 years; 17 had nursed residents with COVID-19. RNs' experiences resonated with Tronto's five tenets of ethical care: attentiveness, responsibility, competence, responsiveness, and solidarity. All nurses described being attentive to needs of others, but were less attentive to their own needs, which came at personal cost. RNs were aware of their professional and leadership responsibilities, being as responsive as they could be to resident needs, processing and sharing rapidly changing guidance and implementing appropriate infection control measures, but felt that relatives and regulatory bodies were not always appreciative. RNs developed enhanced clinical skills, increasing their professional standing, but reported having to compromise care, leading to moral distress. Broadly, participants reported a sense of solidarity across care-home staff and working together to cope with the crisis. Conclusion: Care-home nurses felt unprepared for managing the COVID-19 pandemic, many experienced moral distress. Supporting care-home nurses to recover from the pandemic is essential to maintain a healthy, stable workforce and needs to be specific to care-home RNs, recognizing their unique pandemic experiences. Support for RNs will likely benefit other care-home workers either directly through wider roll-out, or indirectly through improved wellbeing of nurse leaders. Clinical relevance: The COVID-19 pandemic, an international public health emergency, created many challenges for Registered Nurses (RNs) working in long-term care facilities for older people, as residents were particularly vulnerable to the impact of the SARS-CoV-2 virus. Care-home RNs faced challenges distinct from their hospital-based nursing peers and non-nursing social care colleagues due to their isolation, leadership roles, professional legal obligations, and ethical responsibilities, leading to psychological distress on the one hand, but also a newly found confidence in their existing and newly developed skills, and increased recognition by the wider health community of their specialisms