Anticancer,antioxidant and antibiotic activities of mushroom Ramaria flava

Ramaria flava is a species of edible mushroom with some bioactivity. The anticancer, antioxidant and antibiotic activities and chemical composition of R. flava ethanol extract (EE) were evaluated. The present study exhibited that the EE displayed the strongest inhibitory activity against tumor cell MDA-MB-231 with an IC₅₀ value of 66.54 μg/mL in three tested tumor cell lines, and the inhibition percent was 71.66% at the concentration of 200 μg/mL (MTT assay). The total phenolic compounds varied among four fractions of the EE from 6.66 to 61.01 mg gallic acid equivalent (GAE) per g dry weight. Water fraction exhibited high DPPH and OH radical-scavenging activities with low IC₅₀ values of 5.86 and 18.08 μg/mL, respectively. Meanwhile, three phenolic compounds from water fraction were also identified by HPLC. The antibiotic activities of the EE were evaluated against three microorganisms and three fungi strains by means of the agar well diffusion method and the poisoned medium technique, respectively. The EE also showed moderate antibiotic activities. These results suggest that R. flava could hold a good potential source for human health.     

Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells

2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3 mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1–2 mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9–2.3 mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ?34 Mbp. These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines.     

Assessment of antioxidant,anti-inflammatory,anti-cholinesterase and cytotoxic activities of pomegranate (Punica granatum) leaves

This study evaluated antioxidant, anti-inflammatory, anti-cholinesterase and cytotoxic activities of extracts with different polarities (hexane, dichloromethane, ethyl acetate, ethanol and methanol) obtained from Punica granatum leaves. Total phenolics (8.8–127.3 mg gallic acid equivalent/g dry weight), flavonoids (1.2–76.9 mg quercetin equivalent/g dry weight), tannins (63.7–260.8 mg catechin equivalent/kg dry weight) and anthocyanins (0.41–3.73 mg cyanidin-3-glucoside equivalent/g dry weight) of different extracts were evaluated. The methanolic extract presented a good IC50 by DPPH and ABTS assays (5.62 and 1.31 mg/l respectively). The strongest 5-lipoxygenase (5-LOX), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition activities were obtained for the ethanol extract (IC50 values of 6.20, 14.83 and 2.65 mg/l, respectively) and the best cytotoxic activity against MCF-7 cells was obtained for the methanol extract (IC50 = 31 mg/l). These important biological activities showed that P. granatum leaves could be a potential source of the active molecules intended for applications in pharmaceutical industry, but only after additional in vivo experiments.     

Juniperus oxycedrus L. subsp. oxycedrus and Juniperus oxycedrus L. subsp. macrocarpa (Sibth. & Sm.) Ball. “berries” from Turkey: Comparative evaluation of phenolic profile,antioxidant, cytotoxic and antimicrobial activities

This work aimed to evaluate and compare the phenolic profile and some biological properties of the ripe “berries” methanol extracts of Juniperus oxycedrus L. subsp. oxycedrus (Joo) and Juniperus oxycedrus L. subsp. macrocarpa (Sibth. & Sm.) Ball. (Jom) from Turkey. The total phenolic content resulted about 3-fold higher in Jom (17.89 ± 0.23 mg GAE/g extract) than in Joo (5.14 ± 0.06 mg GAE/g extract). The HPLC–DAD–ESI–MS analysis revealed a similar flavonoid fingerprint in Joo and Jom, whereas a difference in their quantitative content was found (4632 μg/g extract and 12644 μg/g extract). In addition, three phenolic acids were detected in Jom only (5765 μg/g extract), and protocatechuic acid was the most abundant one. The antioxidant capacity of the extracts was evaluated by different in vitro assays: in the DPPH and in the TBA tests a stronger activity in Jom was highlighted, while Joo exhibited higher reducing power and metal chelating activity. Joo and Jom did not affect HepG2 cell viability and both extracts resulted virtually non-toxic against Artemia salina. The extracts were also studied for their antimicrobial potential, displaying efficacy against Gram-positive bacteria.     

Effects of red wine intake on human salivary antiradical capacity and total polyphenol content

The protective effects of grape polyphenols have been reported on oral health, though unreasonable alcohol consumption represents a risk factor for developing oral cancer. The possible effects of red wine consumption on salivary antiradical activity were investigated in healthy volunteers for the first time, to the best of our knowledge. Time-course (from 0 min to 240 min) changes of salivary radical-scavenging capacity were measured by the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, in twelve healthy volunteers, after the intake of red wine (125 mL), a capsule of red wine extract (300 mg) or water (125 mL). Furthermore, time-course of salivary total polyphenol levels, detected by the Folin–Ciocalteu colorimetric method, was also determined. Both ABTS and DPPH tests showed that red wine consumption did not increase salivary antiradical activity in volunteers. Conversely, red wine extract administration caused a marked rise in salivary ABTS radical-scavenging capacity within 30 min, followed by a plateau up to 240 min. The same treatment also raised salivary DPPH radical-scavenging activity at any time point, though to a minor extent. The highest salivary polyphenol concentration was reached 30 min after wine drinking, followed by a steady decrease up to 240 min. Wine drinking was not associated to a reduced salivary antiradical capacity. However, wine extract greatly improved the salivary antioxidant status.     

Bioguided isolation of potential antitumor agents from the aerial parts of cultivated cardoon (Cynara cardunculus var. altilis)

Hepatocellular carcinoma (HCC) is one of the leading causes of mortality worldwide; therefore, searching for an effective treatment for this illness is of great importance. In the present work, in vitro cytotoxic activity of the ethanol extract of the aerial parts of Cynara cardunculus L. against human liver carcinoma cells (Hep G2) was tested. Additionally, the antitumor activity of the extract was confirmed using chemically induced rat liver carcinogenesis with diethylnitrosamine (DEN). Moreover, bioguided fractionation and column chromatographic separation of the active compounds were carried out. The extract of C. cardunculus showed a promising cytotoxic activity according to the protocols of the National Cancer Institute. Bioguided chromatographic separation of the ethanol extract of C. cardunculus led to the isolation of seven secondary metabolites including two sesquiterpene lactones as the principal active components of the methylene chloride soluble fraction, grosheimin (IC₅₀ = 7.49 µg/mL) and cynaropicrin (IC₅₀ = 13.9 µg/mL). The compounds were characterized by different spectroscopic techniques such as EI-MS, IR and NMR. Additionally, in silico analysis of the two active compounds revealed their ability to bind with caspase-3 via hydrogen bonds interactions to initiate apoptosis of cancer cells. The results shed the light on the significance of C. cardunculus as a potential source of antitumor agents.     

Folate-mediated poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting drug delivery

A novel targeting drug delivery system (TDDS) has been developed. Such a TDDS was prepared by W₁/O/W₂ solvent extraction/evaporation method, adopting poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] as the drug carrier, folic acid (FA) as the targeting ligand, and doxorubicin (DOX) as the model anticancer drug. The average size, drug loading capacity and encapsulation efficiency of the prepared DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA–PEG–P(HB-HO) NPs) were found to be around 240 nm, 29.6% and 83.5%. The in vitro release profile displayed that nearly 50% DOX was released in the first 5 days. The intracellular uptake tests of the nanoparticles (NPs) in vitro showed that the DOX/FA–PEG–P(HB-HO) NPs were more efficiently taken up by HeLa cells compared to non-folate-mediated P(HB-HO) NPs. In addition, DOX/FA–PEG–P(HB-HO) NPs (IC₅₀ = 0.87 μM) showed greater cytotoxicity to HeLa cells than other treated groups. In vivo anti-tumor activity of the DOX/FA–PEG–P(HB-HO) NPs showed a much better therapeutic efficacy in inhibiting tumor growth, and the final mean tumor volume was 178.91 ± 17.43 mm³, significantly smaller than normal saline control group (542.58 ± 45.19 mm³). All these results have illustrated that our techniques for the preparing of DOX/FA–PEG–P(HB-HO) NPs developed in present work are feasible and these NPs are effective in selective delivery of anticancer drug to the folate receptor-overexpressed cancer cells. The new TDDS may be a competent candidate in application in targeting treatment of cancers.     

NGF induced differentiated PC12 cells as in vitro tool to study 4-hydroxynonenal induced cellular damage

Investigations were carried out to examine the suitability of PC12 cells as an in vitro tool to examine 4-hydroxynonenal (4-HNE)-induced toxicity in nervous tissue. On day 8 of differentiation, markers of neural effects and oxidative stress were measured following exposure of PC12 cells to 1–50 μM 4-HNE for 1–8 h. Endpoints included dopamine DA-D₂ receptor and glutathione S-transferase (GSTP1-1) protein levels, 4-HNE–protein binding, glutathione (GSH) concentrations and intracellular calcium levels. GSH levels were maximally depleted after 4 h. 4-HNE also induced depletion of GSTP1-1 and increased intracellular Ca⁺⁺, with the latter seen as early as 1 h after exposure. Responses at 8 h were not greater than responses at earlier times. The experiments suggest that PC12 cells could be an in vitro tool for understanding toxicant–cell interactions, especially those that result in oxidative stress.     

1-(2-(2-(2-(1-Aminoethyl)phenyl)diselanyl)phenyl)ethanamine: An amino organoselenium compound with interesting antioxidant profile

Free radical scavenging and antioxidant activities of 1-(2-(2-(2-(1-aminoethyl)phenyl)diselanyl)phenyl)ethanamine (compound A) and diphenyl diselenide (PhSe)2 were examined and compared for inhibition of Fe(II) and sodium nitroprusside (SNP) stimulated lipid peroxidation in rat brain, interaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable free radical and their glutathione peroxidase (GPx) like antioxidant activities with H₂O₂ or tBuOOH as substrates and with PhSH as thiol co-substrates as well as their ability to oxidize mono- and di-thiols were also evaluated. This study revealed that an amino group in amino diselenide drastically enhances their catalytic activities in the aromatic thiol (PhSH) assay system. Compound A was ∼2-fold more active than (PhSe)2 in both tBuOOH and H₂O₂ assay systems. In addition, the present results showed that (PhSe)2 exhibited an increased ability to oxidize di-thiols, compound A was not a good substrate for the oxidation of thiol used namely DTT, cystine and DMPS. The antioxidant potency against Fe(II) and SNP-induced brain TBARS were in this order [(compound A); IC₅₀ 2 μM and 4 μM] > [(PhSe)2; IC₅₀ 19 μM and 27.5 μM. Compound A showed DPPH radical-scavenging activity. This study provides in vitro evidence anti-oxidant action of the tested organoselenium compounds, that the nitrogen atom in the organochalcogens can have a profound effect on their antioxidant activity.     

Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress

The protective effect of extract of Crataegus pinnatifida (Rosaceae) pollen (ECPP) on the DNA damage response to oxidative stress was investigated and assessed with an alkaline single-cell gel electrophoresis (SCGE) assay and pBR322 plasmid DNA breaks in site-specific and non-site-specific systems. Total phenolic content, total flavonoid content, individual phenolic compounds, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavenging activity, FRAP, and chelating activity) were also determined. The results showed that ECPP possessed a strong ability to protect DNA from being damaged by hydroxyl radicals in both the site-specific system and the non-site-specific system. It also exhibited a cytoprotection effect in mouse lymphocytes against H₂O₂-induced DNA damage. These protective effects may be related to its high total phenolic content (17.65 ± 0.97 mg GAE/g), total flavonoid content (8.04 ± 0.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.48 ± 0.21 mg Na₂EDTA/g).     

TiO2 nanoparticles induce endothelial cell activation in a pneumocyte–endothelial co-culture model

The effects of particulate matter (PM) on endothelial cells have been evaluated in vitro by exposing isolated endothelial cells to different types of PM. Although some of the findings from these experiments have been corroborated by in vivo studies, an in vitro model that assesses the interaction among different cell types is necessary to achieve more realistic assays. We developed an in vitro model that mimics the alveolar–capillary interface, and we challenged the model using TiO₂ nanoparticles (TiO₂-NPs). Human umbilical endothelial cells (HUVECs) were cultured on the basolateral side of a membrane and pneumocytes (A549) on the apical side. Confluent co-cultures were exposed on the apical side to 10 μg/cm² of TiO₂-NPs or 10 ng/mL of TNFα for 24 h. Unexposed cultures were used as negative controls. We evaluated monocyte adhesion to HUVECs, adhesion molecule expression, nitric oxide concentration and proinflammatory cytokine release. The TiO₂-NPs added to the pneumocytes induced a 3- to 4-fold increase in monocyte adhesion to the HUVECs and significant increases in the expression of adhesion molecules (4-fold for P-selectin at 8 h, and about 8- and 10-fold for E-selectin, ICAM-1, VCAM-1 and PECAM-1 at 24 h). Nitric oxide production also increased significantly (2-fold). These results indicate that exposing pneumocytes to TiO₂-NPs causes endothelial cell activation.     

Ethanolic extract of roots from Arctium lappa L. accelerates the healing of acetic acid-induced gastric ulcer in rats: Involvement of the antioxidant system

We evaluate the curative efficacy of the ethanolic extract (EET) of roots from Arctium lappa (bardana) in healing of chronic gastric ulcers induced by 80% acetic acid in rats and additionally studies the possible mechanisms underlying this action.Oral administration of EET (1, 3, 10 and 30 mg/kg) reduced the gastric lesion area in 29.2%, 41.4%, 59.3% and 38.5%, respectively, and at 10 mg/kg promoted significant regeneration of the gastric mucosa, which was confirmed by proliferating cell nuclear antigen immunohistochemistry. EET (10 mg/kg) treatment did not increase the gastric mucus content but restored the superoxide dismutase activity, prevented the reduction of glutathione levels, reduced lipid hydroperoxides levels, inhibited the myeloperoxidase activity and reduced the microvascular permeability. In addition, EET reduced the free radical generation and increased scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals in vitro. Furthermore, intraduodenal EET (10 and 30 mg/kg) decreased volume and acidity of gastric secretion. Total phenolic compounds were high in EET (Folin–Ciocalteau assay) and the analysis by liquid chromatography-mass spectrometry revealed that the main compounds present in EET were a serie of hydroxycinnamoylquinic acid isomers. In conclusion, these data reveal that EET promotes regeneration of damaged gastric mucosa, probably through its antisecretory and antioxidative mechanisms.     

Chemical composition and anticancer,antiinflammatory, antioxidant and antimalarial activities of leaves essential oil of Cedrelopsis grevei

The essential oil from Cedrelopsis grevei leaves, an aromatic and medicinal plant from Madagascar, is widely used in folk medicine. Essential oil was characterized by GC–MS and quantified by GC–FID. Sixty-four components were identified. The major constituents were: (E)-β-farnesene (27.61%), δ-cadinene (14.48%), α-copaene (7.65%) and β-elemene (6.96%). The essential oil contained a complex mixture consisting mainly sesquiterpene hydrocarbons (83.42%) and generally sesquiterpenes (98.91%). The essential oil was tested cytotoxic (on human breast cancer cells MCF-7), antimalarial (Plasmodium falciparum), antiinflammatory and antioxidant (ABTS and DPPH assays) activities. C. grevei essential oil was active against MCF-7 cell lines (IC₅₀ = 21.5 mg/L), against P. falciparum, (IC₅₀ = 17.5 mg/L) and antiinflammatory (IC₅₀ = 21.33 mg/L). The essential oil exhibited poor antioxidant activity against DPPH (IC₅₀ > 1000 mg/L) and ABTS (IC₅₀ = 110 mg/L) assays. A bibliographical review was carried out of all essential oils identified and tested with respect to antiplasmodial, anticancer and antiinflammatory activities. The aim was to establish correlations between the identified compounds and their biological activities (antiplasmodial, anticancer and antiinflammatory). According to the obtained correlations, 1,4-cadinadiene (R² = 0.61) presented a higher relationship with antimalarial activity. However, only (Z)-β-farnesene (R² = 0.73) showed a significant correlation for anticancer activity.     

Leaf extracts from Teucrium ramosissimum protect against DNA damage in human lymphoblast cell K562 and enhance antioxidant,antigenotoxic and antiproliferative activity

The in vitro antioxidant, antigenotoxic and antiproliferative activities of Teucrium ramosissimum extracts were investigated. The antioxidant activities of the tested extracts were evaluated through three chemical assays: The Cupric reducing antioxidant capacity, the reducing power and the ferric reducing antioxidant power. TR1 fraction from methanol extract showed the best antioxidant activity evaluated by the CUPRAC, RP and FRAP assays with TEAC values of 4.04, 1.77 and 1.48 μM respectively compared to control. Yet, TR2 fraction exhibited the lowest antioxidant effect with a TEAC values of 1.97, 0.408 and 0.35 μM respectively. All the tested extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals. Furthermore, the effects of T. ramosissimum extracts on cell proliferation were also examined. The cytotoxic study revealed that methanol extract significantly inhibited the proliferation of K562 cells (IC50 = 150 μg/mL). The antigenotoxic properties of these extracts were investigated by assessing the induction and inhibition of the genotoxicity induced by the direct-acting mutagen, hydrogen peroxide (H₂O₂), using an eukaryotic system; the “Comet assay.” The results showed that all the extracts inhibited the genotoxicity induced by H₂O₂, and particularly TR2 fraction (96.99%) and methanol extract (96.64%).The present study has demonstrated that T. ramosissimum extract possess potent antioxidant, antiproliferative and antigenotoxic activities, which could be derived from compounds such as flavonoids and polyphenols.     

Antioxidant activity of a novel synthetic hexa-peptide derived from an enzymatic hydrolysate of duck skin by-products

A peptide was synthesized on the basis of our previous study from solid phase peptide synthesis using ASP48S (Peptron Inc.) and identified by the reverse phase high-performance liquid chromatography (HPLC) using a Vydac Everest C₁₈ column. The molecular mass of the peptide found to be 693.90 Da, and the amino acid sequences of the peptide was Trp–Tyr–Pro–Ala–Ala–Pro. The purpose of this study was to evaluate antioxidant effects of the peptide by electron spin resonance (ESR) spectrometer, and on t-BHP-induced liver cells damage in Chang cells. The antioxidative activity of the peptide was evaluated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, alkyl and superoxide radical scavenging activity using an ESR spectrometer. The half maximal inhibitory concentration (IC₅₀) value of the peptide for hydroxyl, DPPH, alkyl, and superoxide radical scavenging activity were 45.2, 18.5, 31.5, and 33.4 μM, respectively. In addition, the peptide inhibited productions of cell death against t-BHP-induced liver cell damage in Chang cells. It was presumed to be peptide involved in regulating the apoptosis-related gene expression in the cell environment. The present results indicate that the peptide substantially contributes to antioxidative properties in liver cells.     

Activation of human neutrophils by titanium dioxide (TiO2) nanoparticles

This paper describes the in vitro effects of titanium dioxide (TiO₂) nanoparticles (NPs) upon human neutrophils. Kinetic experiments revealed no cell necrosis after 24 h of treatment with TiO₂ (0–100 μg/ml). In contrast, TiO₂-induced change in cellular morphology in a concentration-dependent manner in neutrophils over time, indicating its potential to activate these cells. To further support this, we demonstrated that TiO₂ markedly and rapidly induced tyrosine phosphorylation events, including phosphorylation of two key enzymes, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases-1/2 (Erk-1/2). We also determined the effects of TiO₂ on two neutrophil functions requiring a longer exposure period between NPs and cells: apoptosis and cytokine production. Interestingly, at concentrations ⩾20 μg/ml, TiO₂ inhibited neutrophil apoptosis in a concentration-dependent manner after 24 h of treatment. Supernatants from TiO₂-induced neutrophils were harvested after 24 h and tested for the presence of 36 different analytes (cytokines, chemokines) using an antibody array assay. TiO₂ treatment increased production of 13 (36%) analytes, including IL-8, which exhibited the greatest increase (∼16 × control cell levels). The increased production of IL-8 was confirmed by ELISA. We conclude that TiO₂ exerts important neutrophil agonistic properties in vitro.     

Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC₅₀: 11 μM; 48 h IC₅₀: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC₅₀: 47 μM; 48 h IC₅₀: 17 μM). IC₅₀ values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC₅₀ values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7–123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.     

Potential anti-inflammatory effect of Leea macrophylla Roxb. leaves: A wild edible plant

Leea macrophylla (Leeaceae) is a wild edible plant with ethomedicinal importance as anti-inflammatory agent. However, no systematic studies on its anti-inflammatory activity and mechanisms have been reported. Present study was undertaken to evaluate anti-inflammatory activity of methanol extract of L. macrophylla leaves. Phytochemical investigation revealed presence of sterols, triterpenoids and ascorbic acid in extract. Methanol extract inhibited lipopolysaccharide stimulated production of inflammatory mediators viz. prostaglandin E2, tumor necrotic factor-α, interleukin-6 and interleukin-1β in vitro in mouse peritoneal macrophages. Additionally, the in vivo anti-inflammatory activity of this extract was evaluated by using carrageenan induced paw edema and cotton pellet granuloma assays in experimental rats. Oral administration of extract (100 and 200 mg/kg) exhibited dose dependant inhibition of carrageenan induced inflammation (p < 0.05) and the reduction of the granuloma tissue formation (p < 0.05–0.01). The extract (100 and 200 mg/kg, orally) exhibited significant central and peripheral analgesic activity in hot-plate test (p < 0.01) and acetic acid induced writhing test (p < 0.05–0.01) respectively in experimental mice. Treatment with extract (100 and 200 mg/kg, orally) significantly reduced the yeast provoked elevated body temperature (p < 0.05–0.01) in experimental rats. These results confirmed the traditional anti-inflammatory indication of L. macrophylla leaves.     

Safety studies on products from whole coffee fruit

The fruit of the coffee plant, Coffea arabica, has high phenolic antioxidant and phytonutrient content and could be a beneficial food ingredient. However, the fruit has historically been discarded for the favored harvesting of the coffee bean alone. CoffeeBerry® products are derived from the whole fruit and include a ground whole powder, a water extract, and a more recently developed water–ethanol extract. The safety of CoffeeBerry® products was evaluated in three genotoxicity studies, three short-term oral toxicity studies, and a 90-day dietary toxicity study. Bacterial mutagenicity studies and a micronucleus test using murine peripheral cells demonstrated that none of the three products showed mutagenic or genotoxic potential. In the short-term studies, despite palatability issues, female rats showed a tolerance for whole powder and ethanol extract at doses up to 8800 mg/kg bw/day. Male rats also exhibited palatability issues and tolerated lower doses of approximately 4000 mg/kg bw/day ethanol extract via gavage and approximately 2100 mg/kg bw/day whole powder or water extract in the diet. When fed in the diet to Sprague–Dawley rats for 90 days, ethanol extract showed no adverse effects at dietary concentrations of up to 5% (approximately 3446 and 4087 mg/kg bw/day for male and female rats, respectively).     

Oil-Frozen W1/O/W2 Double Emulsions for Dermal Biomacromolecular Delivery Containing Ethanol as Chemical Penetration Enhancer

Oil-frozen water-in-oil-in-water (W₁/O/W₂) double emulsions (DE) containing ethanol up to 40% (w/v) in the external aqueous W₂ phase exhibited external coalescence upon thawing of the oil phase, releasing up to 85% of the encapsulated protein of the internal aqueous phase. These emulsions were studied in vitro as potential dermal macromolecular delivery formulations, achieving fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) penetration of up to 86 μm into porcine skin, reaching the viable epidermis where the immunocompetent Langerhans cells are located. Enzyme-linked immunosorbent assay was performed to observe the effect of the emulsification process and ethanol content on the ability of BSA to form antigen–antibody complexes; results indicated that ethanol content and the emulsification process did not diminish the BSA–antibody complex formation when compared with a BSA standard aqueous solution. Therefore, it is shown that oil-frozen W₁/O/W₂ DE, with penetration-enhancing ethanol in the W₂ phase, can potentially be used for cutaneous vaccine delivery formulations.