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.

C3H Expression Is Crucial for Methyl Jasmonate Induction of Chicoric Acid Production by Echinacea purpurea (L.) Moench Cell Suspension Cultures.

Echinacea purpurea (L.) Moench is one of the most economically important medicinal plants, cultivated worldwide for its high medicinal value and with several industrial applications in both pharmaceutical and food industries. Thanks to its various phytochemical contents, including caffeic acid derivatives (CADs), E. purpurea extracts have antioxidant, anti-inflammatory, and immuno-stimulating properties. Among CADs, chicoric acid is one of the most important compounds which have shown important pharmacological properties. The present research was aimed at optimizing the production of chicoric acid in E. purpurea cell culture. Methyl jasmonate (MeJa) at different concentrations and for different duration of treatments was utilized as elicitor, and the content of total polyphenols and chicoric acid was measured. Several genes involved in the chicoric acid biosynthetic pathway were selected, and their expression evaluated at different time points of cell culture growth. This was performed with the aim of identifying the most suitable putative molecular markers to be used as a proxy for the early prediction of chicoric acid contents, without the need of expensive quantification methods. A correlation between the production of chicoric acid in response to MeJa and an increased response to oxidative stress was also proposed.

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.

Phenylpropanoid glycosides from plant cell cultures induce heme oxygenase 1 gene expression in a human keratinocyte cell line by affecting the balance of NRF2 and BACH1 transcription factors.

Phenylpropanoids have several highly significant biological properties in both plants and animals. Four phenylpropanoid glycosides (PPGs), verbascoside (VB), forsythoside B (FB), echinacoside (EC) and campneoside I (CP), were purified and tested for their capability to activate NRF2 and induce phase II cytoprotective enzymes in a human keratinocyte cell line (HaCaT). All four substances showed similar strong antioxidant and radical-scavenging activities as determined by diphenylpicrylhydrazyl assay. Furthermore, in HaCaT cells, FB and EC are strong activators of NRF2, the nuclear transcription factor regulating many phase II detoxifying and cytoprotective enzymes, such as heme oxygenase 1 (HMOX1). In HaCaT cells, FB and EC (200 µM) induced nuclear translocation of NRF2 protein after 24 h and reduced nuclear protein levels of BACH1, a repressor of the antioxidant response element. FB and EC greatly HMOX1 mRNA levels by more than 40-fold in 72 h. Cytoplasmic HMOX1 protein levels were also increased at 48 h after treatment. VB was less active compared to FB and EC, and CP was slightly active only at later times of treatment. We suggest that hydroxytyrosol (HYD) could be a potential bioactive metabolite of PPGs since HYD, in equimolar amounts to PGGs, is able to both activate HO-1 transcription and modify Nrf2/Bach1 nuclear protein levels. This is in agreement with the poor activity of CP, which contains a HYD moiety modified by an O-methyl group. In conclusion, FB and EC from plant cell cultures may provide long-lasting skin protection by induction of phase II cytoprotective capabilities.

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.

Irbic acid, a dicaffeoylquinic acid derivative from Centella asiatica cell cultures.

3,5-O-dicaffeoyl-4-O-malonilquinic acid (1) (irbic acid) has been isolated for the first time from cell cultures of Centella asiatica and till now it has never been reported to be present in the intact plant. Evidence of its structure was obtained by spectroscopic analyses (MS/NMR). Besides 1, cell cultures produce also the known 3,5-O-dicaffeoylquinic acid, chlorogenic acid, and the triferulic acid 2 (4-O-8'/4'-O-8″-didehydrotriferulic acid). Biological activities were evaluated for compound 1, which showed to have a strong radical scavenging capacity, together with a high inhibitory activity on collagenase. This suggests a possible utilization of this substance as a topical agent to reduce the skin ageing process.

Effects of verbascoside, biotechnologically purified by Syringa vulgaris plant cell cultures, in a rodent model of periodontitis.

OBJECTIVES: Verbascoside has previously been characterized as an effective scavenger of active free radicals and an inhibitor of lipid peroxidation. In the present study, we have investigated the effects of verbascoside from Syringa vulgaris in a rat model of ligature-induced periodontitis. METHODS: Male Sprague-Dawley rats were lightly anaesthetized with pentobarbitone (35 mg/kg). Sterile, 2-0 black braided silk thread was placed around the cervix of the lower left first molar and knotted medially. Animals received vebascoside 2 mg/kg orally, daily for 8 days. KEY FINDINGS: On the eighth day after placement of the ligature, we evaluated several markers of inflammation: (i) myeloperoxidase activity, (ii) thiobarbituric acid-reactant substance measurements, (iii) NF-κB expression, (iv) iNOS expression, (v) the nitration of tyrosine residues, (vi) activation of the nuclear enzyme poly(ADP-ribose) polymerase, (vii) Bax and Bcl-2 expression and (viii) a degree of gingivomucosal tissue injury. Oral administration of verbascoside (2 mg/kg daily for 8 days) significantly decreased all of the parameters of inflammation as described above. CONCLUSIONS: These results demonstrate that verbascoside exerts an anti-inflammatory role during experimental periodontitis and is able to ameliorate the tissue damage associated with ligature-induced periodontitis.

PPAR-alpha Contributes to the Anti-Inflammatory Activity of Verbascoside in a Model of Inflammatory Bowel Disease in Mice.

The previous results suggest that peroxisome proliferator-activated receptor-alpha (PPAR)-alpha, an intracellular transcription factor activated by fatty acids, plays a role in control of inflammation. There is persuasive epidemiological and experimental evidence that dietary polyphenols have anti-inflammatory activity. In this regard, it has been demonstrated that verbascoside (VB) functions as intracellular radical scavenger and reduces the microscopic and macroscopic signs of experimental colitis. With the aim to characterize the role of PPAR-alpha in VB-mediated anti-inflammatory activity, we tested the efficacy of VB in an experimental model of inflammatory bowel disease induced by dinitrobenzene sulfonic acid, comparing mice lacking PPAR-alpha (PPAR-alphaKO) with wild type (WT) mice. Results indicate that VB-mediated anti-inflammatory activity is weakened in PPAR-alphaKO mice, compared to WT controls, especially in the inhibition of neutrophil infiltration, intestinal permeability and colon injury. These results indicate that PPAR-alpha can contribute to the anti-inflammatory activity of VB in inflammatory bowel disease.

Protective effect of verbascoside in activated C6 glioma cells: possible molecular mechanisms.

The glycosylated phenylpropanoid verbascoside (VB), isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae), has previously been characterized as an effective scavenger of biologically active free radicals and an inhibitor of lipid peroxidation. The aim of the present study was to evaluate in a rat glioma cell line (C6) the effect of VB biotechnologically produced by S. vulgaris plant cell cultures in the regulation of the inflammatory response. We used a model of central nervous system inflammation induced by bacterial endotoxin/cytokine (lipopolysaccharide (LPS)/interferon (IFN)-gamma, 1 microg/ml and 100 U/ml, respectively). Our results show that the treatment with LPS/IFN-gamma for 24 h elicited the induction of inducible nitric oxide synthase (iNOS) activity as determined by NO(x) accumulation in the culture medium. Preincubation with VB (10-100 microg/ml) abrogated the mixed cytokine-mediated induction of iNOS. The effect was concentration-dependent. Our studies also showed an inhibitory effect of VB on neuronal nitric oxide synthase expression. Moreover, Western blot analysis showed that this glycoside prevents specifically the activation of the proinflammatory enzyme cyclooxygenase (COX)-2 in glioma cells without simultaneous inhibition of COX-1 enzyme. Moreover, we found that VB reduced the expression of proinflammatory enzymes in LPS/IFN-gamma through the inhibition of the activation of nuclear factor kappa B and mitogen-activated protein kinase signaling pathway. The mechanisms underlying in vitro the neuroprotective properties of VB involve modulation of transcription factors and consequent altered gene expression, resulting in downregulation of inflammation. These findings provide support that VB may provide a promising approach for the treatment of oxidative-stress-related neurodegenerative diseases.

3,5-Dicaffeoyl-4-malonylquinic acid reduced oxidative stress and inflammation in a experimental model of inflammatory bowel disease.

AIM: The aim of the present study was to examine the effects of 3,5-dicaffeoyl-4-malonylquinic acid (CA1), extract from Centella Asiatica, in rats subjected to experimental colitis. RESULTS: Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). CA1 was administered daily orally (0.2 or 2 mg/kg). Four days after DNBS administration, treatment with CA1 significantly reduced the appearance of diarrhoea and the loss of body weight. This was associated with a significant reduction in colonic MPO activity. CA1 also reduced NF-kappaB activation, the pro-inflammatory cytokines release, the appearance of I-NOS, nitrotyrosine, PARP and proMMP-9 and -2 activity in the colon and reduced the up-regulation of ICAM-1 and the expression of P-Selectin. CONCLUSIONS: The results of this study suggested that administration of CA1 may be beneficial for treatment of inflammatory bowel disease.

Effects of verbascoside biotechnologically produced by Syringa vulgaris plant cell cultures in a rodent model of colitis.

The aim of the present study was to examine the effects of verbascoside (VB) in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of 2,4 dinitrobenzene sulfonic acid (DNBS; 25 mg/rat). VB was administered daily per os (0.2 and 2 mg/kg) 4 days after DNBS administration in the colon. Treatment with VB significantly (P < 0.01) reduced macroscopic damage score, loss of body weight, myeloperoxidase activity and thiobarbituric acid-reactant substances. Moreover, the intensity of the positive staining for tumor necrosis factor-alpha, interleukin-1beta, intercellular adhesion molecule-1, P-selectin, inducible nitric oxide synthase, and poly(ADP ribose) was also significantly (P < 0.01) reduced by VB treatment. Therefore, VB treatment significantly (P < 0.01) reduced the degree of NF-kappaB p65 and activation of the pro-active form metalloproteinase (MMP)-2 and pro-MMP-9 activity. The results of this study suggested that VB functions as an intracellular radical scavenger and so reduces the microscopic and macroscopic signs of colitis in the rat. Therefore, administration of VB may be beneficial for the treatment of inflammatory bowel disease.

Teupolioside, a phenylpropanoid glycosides of Ajuga reptans, biotechnologically produced by IRBN22 plant cell line, exerts beneficial effects on a rodent model of colitis.

The aim of the present study was to examine the effects of phenylpropanoid glycoside, teupolioside, biotechnologically produced by IRBN22 Ajuga reptans cell line, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Teupolioside was administered daily orally (0.2 or 2mgkg(-1)). On Day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. Four days after DNBS administration, colon TNF-alpha and IL-1beta productions were increased, associated with colon damage. Neutrophil infiltration, by myeloperoxidase activity, in the mucosa was associated with up-regulation of ICAM-1 and P-selectin and high levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) polymerase (PARP) showed an intense staining in the inflamed colon. Biochemical methods and zymography were used to analyze MMP-9 and -2 activities in colon tissues from DNBS-injured rats. Treatment with teupolioside significantly reduced the appearance of diarrhoea and the loss of body weight. This was associated with a remarkable amelioration in the disruption of the colonic architecture and a significant reduction in colonic myeloperoxidase activity and malondialdehyde levels. Teupolioside also reduced the pro-inflammatory cytokines release, the appearance of nitrotyrosine and PARP immunoreactivity in the colon and reduced the up-regulation of ICAM-1 and the expression of P-selectin. Therefore, teupolioside also reduced proMMP-9 and -2 activity induced in the colon by DNBS administration. The results of this study suggested that administration of teupolioside may be beneficial for treatment of inflammatory bowel disease.

Plant polyphenols against UV-C-induced cellular death.

The glycosylated phenylpropanoid verbascoside isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae) has previously been characterized as an effective scavenger of biologically active free radicals such as hydroxyl, superoxide, and nitric oxide, as a chelator of redox active transition metal ions (Fe (2+), Fe (3+), Cu (2+), and Ni (2+)), and an inhibitor of lipid peroxidation. In the present work, we have compared the cytoprotective effects of the biotechnologically produced verbascoside with two commercially available polyphenols (the glycosylated flavonoid rutin and its aglycone quercetin) against free radical-mediated UVC-induced cellular death in cultures of human keratinocytes (HaCaT) and breast cancer cells (MCF 7). We have shown that all the polyphenols studied afforded effective protection against UVC-induced necrosis and did not prevent UVC-induced apoptosis in both normal and tumor cell lines. The cytoprotection did not correlate either with UVC absorbance by polyphenols or with their superoxide radical scavenging properties. However, UVC protection strongly depended on the lipid peroxidation inhibiting and Fe (2+) chelating properties of polyphenols. We suggest that these plant polyphenols could be feasible for a photoprotection of human skin.