A Novel Perilla frutescens (L.) Britton Cell-Derived Phytocomplex Regulates Keratinocytes Inflammatory Cascade and Barrier Function and Preserves Vaginal Mucosal Integrity In Vivo.

In the last years, the medicinal plant Perilla frutescens (L.) Britton has gained scientific interest because leaf extracts, due to the presence of rosmarinic acid and other polyphenols, have shown anti-allergic and skin protective potential in pre-clinical studies. Nevertheless, the lack of standardized extracts has limited clinical applications to date. In this work, for the first time, a standardized phytocomplex of P. frutescens, enriched in rosmarinic acid and total polyphenols, was produced through innovative in vitro cell culture biotechnology and tested. The activity of perilla was evaluated in an in vitro inflammatory model of human keratinocytes (HaCaT) by monitoring tight junctions, filaggrin, and loricrin protein levels, the release of pro-inflammatory cytokines and JNK MAPK signaling. In a practical health care application, the perilla biotechnological phytocomplex was tested in a multilayer model of vaginal mucosa, and then, in a preliminary clinical observation to explore its capacity to preserve vaginal mucosal integrity in women in peri-menopause. In keratinocytes cells, perilla phytocomplex demonstrated to exert a marked activity in epidermis barrier maintenance and anti-inflammatory effects, preserving tight junction expression and downregulating cytokines release through targeting JNK activation. Furthermore, perilla showed positive effects in retaining vaginal mucosal integrity in the reconstructed vaginal mucosa model and in vivo tests. Overall, our data suggest that the biotechnological P. frutescens phytocomplex could represent an innovative ingredient for dermatological applications.

A Phytocomplex Obtained from Salvia officinalis by Cell Culture Technology Effectively Controls the Grapevine Downy Mildew Pathogen Plasmopara viticola.

The negative impact of using conventional fungicides in plant disease protection has increased the interest in safer alternatives such as plant secondary metabolites, generally having a better toxicological profile. However, cultivation conditions and plant material strongly affect the quality and quantity of secondary metabolites obtained from field grown plants, limiting the standardization needed for industrial production. Plant cell culture technology can provide highly homogeneous biomasses with specific chemical characteristics. A phytocomplex with high rosmarinic acid content (10.12% w/w) was obtained from a selected cell line of Salvia officinalis and was tested against the grapevine downy mildew pathogen, Plasmopara viticola. Grapevine leaf discs were sprayed with the phytocomplex at 5 g/L and then inoculated with P. viticola sporangia. Sporulation level on each disc was assessed after 7 days with an image processing software. The phytocomplex reduced by 95% the sporulation level compared to the control and was also more effective than rosmarinic acid alone, used at the same concentration found in the phytocomplex. Persistence of the phytocomplex was also assessed: when applied 5 days before inoculation, it reduced by 90% the sporulation level compared to the control. These results highlight the possibility to take advantage of cell culture techniques to produce safer pesticides with high quality standards.

Attenuation of neuroinflammation in microglia cells by extracts with high content of rosmarinic acid from in vitro cultured Melissa officinalis L. cells.

Plant cell culture is a biotechnology cultivation method that permit to cultivate plants in a short period of time and to obtain extracts with a high degree of standardization and high safety profile. The aim of our study was to evaluate the anti-inflammatory and neuroprotective activity of a standardized Melissa officinalis L. phytocomplex extract (MD) obtained with an in vitro plant cell culture. The MD has been chemically characterized and the content of total polyphenols was 5.17 ± 0.1 % w/w, with a content of rosmarinic acid (RA), its main constituent, of 4.02 ± 0.1 % w/w. MD was tested in an in vitro model of neuroinflammation, in which microglia cells (BV2) were stimulated with Lipopolysaccharides (LPS; 250 ng/mL) for 24 h and its pharmacological activity was compared with that of RA. MD (10 µg/mL) and RA (0.4 µg/mL) reduced pro-inflammatory factors (NF-kB, HDAC, IL-1ß) in LPS-stimulated BV2 cells and counteracted the toxic effect produced by activated microglia medium on neuronal cells. This work shows the efficacy of MD on reducing microglia-mediated neuroinflammation and promoting neuroprotection, highlighting the innovative use of in vitro plant cell cultures to obtain contaminant-free extracts endowed with marked activity and improved quali-quantitative ratio in the constituents' content.

Rosa chinensis in vitro cell cultures: a phytocomplex rich of medium molecular weight polysaccharides with hydrating properties.

In-vitro cell cultures of selected Rosa chinensis meristematic cells cultivated with an innovative CROP® (Controlled Release of Optimized Plants) platform, allowed obtaining a stable and standardized phytocomplex rich of medium molecular weight polysaccharides. The polysaccharides profile of the rose extract has been analysed with the size exclusion chromatography (HPLC-ELSD-SEC) both in the in vitro extract and in the dried petals of Rosa chinensis. The polysaccharides content in the extract was ≥20%, higher than in the dried petals. The 65-80% of total polysaccharides have a medium molecular weight (1000 Da), known for their moisturizing and anti-age properties. Reconstructed human epidermis in homeostatic conditions was used to evaluate its moisturizing action and the ability to maintain homeostasis. The Rosa chinensis extract increased the Aquaporin-3 expression and cell membrane localization and demonstrated to regulate hydration either in topical and systemic exposure.

Red Carrot Cells Cultured in vitro Are Effective, Stable, and Safe Ingredients for Skin Care, Nutraceutical, and Food Applications.

Plant biomasses growing in bioreactor could be developed as production systems for cosmetic ingredients, nutraceuticals and food additives. We previously reported that the red carrot cell line R4G accumulates high levels of anthocyanins, which are potent antioxidants with multiple health-promoting properties. To investigate the industrial potential of this cell line in detail, we tested extract for antioxidant and anti-inflammatory activity in the mouse monocyte/macrophage cell-line J774A.1 and in reconstructed skin tissue models. We also compared the R4G extract to commercial carrot extracts in terms of stability and metabolomic profiles. We found that the R4G extract have potent antioxidant and anti-inflammatory activities, protecting mammalian cells from the oxidative stress triggered by exposure to bacterial lipopolysaccharides and H2O2. The extract also inhibited the nuclear translocation of NF-κB in an epidermal skin model, and induced the expression of VEGF-A to promote the microcirculation in a dermal microtissue model. The anthocyanins extracted from R4G cells were significantly more stable than those found in natural red carrot extracts. Finally, we showed that R4G extract has similar metabolomic profile of natural extracts by using a combination of targeted and untargeted metabolomics analysis, demonstrating the safety of R4G carrot cells for applications in the nutraceutical and food/feed industries.

Disorders of consciousness and N400 ERP measures in response to a semantic task.

Disorders of consciousness (DOC) have been studied in recent years. In the present research, electrophysiological measures (ERPs) were used to verify the preservation of semantic linguistic processes in vegetative states (VS) and minimal-consciousness state (MCS). Eighteen patients classified as VS or MCS, and 20 controls were submitted to a semantic associative task with congruous or incongruous word sequences (auditory stimuli). An increased N400 peak amplitude within the fronto-central cortical areas was shown in response to incongruous sequences for both patients and controls. Thus, semantic processing was partially preserved in both VS and MCS patients.

Metabolomic analysis reveals that the accumulation of specific secondary metabolites in Echinacea angustifolia cells cultured in vitro can be controlled by light.

Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14 days) and then compared the metabolomes of dark-grown and illuminated cells by liquid chromatography-mass spectrometry. Among 256 signals, we putatively identified 159 molecules corresponding to 56 different metabolites plus their fragments, adducts and isotopologs. The E. angustifolia metabolome consisted mainly of caffeic acid derivatives, comprising (a) caffeic acid conjugated with tartaric, quinic and hexaric acids; and (b) caffeic acid conjugated with hydroxytyrosol glycosides (e.g., echinacoside, verbascoside and related molecules). Many of these metabolites have not been previously described in E. angustifolia, which currently lacks detailed metabolic profiles. Exposure to light significantly increased the levels of certain caffeic acid derivatives (particularly caffeoylquinic acids and hydroxytyrosol derivatives lacking rhamnose residues) and reduced the level of hydroxytyrosol derivatives with rhamnose residues, revealing that light specifically inhibits the rhamnosylation of caffeoyl phenylethanoid glycosides. These results are significant because they suggest that the metabolic profile of cell cultures can be manipulated by controlling simple environmental variables such as illumination to modulate the levels of potentially therapeutic compounds.

Prevalence and comorbidity of prolonged grief disorder in a sample of caregivers of patients in a vegetative state.

Previous studies have shown that Prolonged Grief Disorder (PGD), Post-traumatic Stress Disorder (PTSD) and major depression are autonomous nosological entities. The present study aims at further analyzing the relationship among them in a sample of caregivers of patients in Vegetative State (VS) or Minimally Conscious State (MCS). We also investigated factors predicting the development of PGD. We sampled 40 Caregivers of patients in VS or MCS consecutively admitted to long-term care units. Caregivers were administered the PG-12, the Depression Questionnaire, the SCID I and the Davidson Trauma Scale. Six participants (15%) fulfilled the criteria for PGD, 25% (N = 10) for depression and 25% (N = 10) for PTSD. Although significant correlations emerged among symptom domains of the three disorders, no relevant association was found between a diagnosis of PGD, depression and PTSD. The severity of PTSD symptoms was found to be predictive of PGD. Clinical implications are discussed.

Prolonged grief and depression in caregivers of patients in vegetative state.

PRIMARY OBJECTIVE: Several studies have shown that the symptoms of grief are different from symptoms of depression among bereaved family members. The present study is an attempt to replicate these findings among caregivers of patients in a vegetative state. METHODS AND PROCEDURES: Forty-five caregivers of 41 patients in vegetative or minimally conscious state in-hospital for long-term care. The questionnaire Prolonged Grief 12 (PG-12) assessed the presence of Prolonged Grief Disorder; the Depression Questionnaire (DQ) assessed the presence of a clinically significant depressive state. MAIN OUTCOMES AND RESULTS: Three conditions of psychiatric illness emerged: Prolonged Grief Disorder only (n = 9; 20%), depression only (n = 7; 15.5%) and Prolonged Grief Disorder together with depression (n = 7; 15.5%). There was no association (V = 0.203) between diagnosis of Prolonged Grief Disorder and the presence of a clinically significant depressive state. Caregiver's syndromal-level grief was associated with patients' young age (p = 0.028) and with younger age of caregiver (p = 0.029). Caregiver's syndromal-level depression was associated with less time from the event (p = 0.003). CONCLUSIONS: Caregiver's grief symptoms are distinct from their depressive symptoms among family members and each disorder has distinct risk factors.

Medicago truncatula ENOD40-1 and ENOD40-2 are both involved in nodule initiation and bacteroid development.

The establishment of a nitrogen-fixing root nodule on legumes requires the induction of mitotic activity of cortical cells leading to the formation of the nodule primordium and the infection process by which the bacteria enter this primordium. Several genes are up-regulated during these processes, among them ENOD40. Here it is shown, by using gene-specific knock-down of the two Medicago truncatula ENOD40 genes, that both genes are involved in nodule initiation. Further, during nodule development, both genes are essential for bacteroid development.