Suppression of lipopolysaccharide-stimulated cytokine/chemokine production in skin cells by sandalwood oils and purified α-santalol and ß-santalol.

Medicinally, sandalwood oil (SO) has been attributed with antiinflammatory properties; however, mechanism(s) for this activity have not been elucidated. To examine how SOs affect inflammation, cytokine antibody arrays and enzyme-linked immunosorbent assays were used to assess changes in production of cytokines and chemokines by co-cultured human dermal fibroblasts and neo-epidermal keratinocytes exposed to lipopolysaccharides and SOs from Western Australian and East Indian sandalwood trees or to the primary SO components, α-santalol and ß-santalol. Lipopolysaccharides stimulated the release of 26 cytokines and chemokines, 20 of which were substantially suppressed by simultaneous exposure to either of the two sandalwood essential oils and to ibuprofen. The increased activity of East Indian SO correlated with increased santalol concentrations. Purified α-santalol and ß-santalol equivalently suppressed production of five indicator cytokines/chemokines at concentrations proportional to the santalol concentrations of the oils. Purified α-santalol and ß-santalol also suppressed lipopolysaccharide-induced production of the arachidonic acid metabolites, prostaglandin E2, and thromboxane B2, by the skin cell co-cultures. The ability of SOs to mimic ibuprofen non-steroidal antiinflammatory drugs that act by inhibiting cyclooxygenases suggests a possible mechanism for the observed antiinflammatory properties of topically applied SOs and provides a rationale for use in products requiring antiinflammatory effects.

The potential use of Echinacea in acne: control of Propionibacterium acnes growth and inflammation.

Acne is a chronic inflammatory disorder of skin follicles caused by the gram-positive bacterium Propionibacterium acnes. The possibility was investigated that a standardized preparation of Echinacea purpurea (Echinaforce®), with known antiviral, antiinflammatory and antibacterial properties, might provide a useful alternative treatment in the control of the disease. The herbal extract readily killed a standard laboratory strain of the bacterium and several clinical isolates. In cell culture models of human bronchial epithelial cells and skin fibroblasts, P. acne induced the secretion of substantial amounts of several pro-inflammatory cytokines, including IL-6 and IL-8 (CXCL8), as determined by means of cytokine-antibody arrays. However, the E. purpurea completely reversed this effect and brought the cytokine levels back to normal. Thus Echinaforce® could provide a safe two-fold benefit to acne individuals by inhibiting proliferation of the organism and reversing the bacterial-induced inflammation.

The efficacy of Echinacea in a 3-D tissue model of human airway epithelium.

We evaluated the antirhinovirus efficacy of a standardized preparation of Echinacea purpurea (Echinaforce) in a 3-dimensional organotypic model of normal human airway epithelium (EpiAirway tissue). Individual replicate tissue samples, maintained as inserts in culture for 3 days or 3 weeks, were infected with rhinovirus type 1A (RV1A), Echinacea alone, a combination of the two, or medium only. None of the treatments affected the histological appearance or integrity of the tissues, all of which maintained a high level of cell viability and preservation of cilia. RV infection resulted in increased mucopolysaccharide inclusions in the goblet cells, but this feature was reversed by Echinacea treatment. This result was confirmed by measurements of mucin secretion, which was stimulated by RV but reversed by Echinacea, suggesting that mucus production during colds could be ameliorated by Echinacea. We did not find evidence of virus replication, although the RV-infected tissues secreted substantial amounts of the pro-inflammatory cytokines IL-6 and IL-8 (CXCL8), and this response was reversed by Echinacea treatment. These results confirmed previous findings derived from studies of bronchial and lung epithelial cell lines, namely, that RV infection results in a substantial inflammatory response in the absence of virus replication.

Bactericidal and anti-inflammatory properties of a standardized Echinacea extract (Echinaforce): dual actions against respiratory bacteria.

Common symptoms of upper respiratory infections, such as sore throat, cough, and inflammation, are often caused by bacteria, sometimes as a complication of virus infection. Extracts of Echinacea purpurea (Asteraceae) have been advocated traditionally for use by individuals suffering from these symptoms, although the underlying basis for the beneficial effects of Echinacea is not known. We hypothesized that Echinacea could inactivate certain respiratory bacteria and could also reverse inflammatory effects caused by these bacteria in epithelial cells. In order to test this we used a commercial standardized extract of Echinacea purpurea (Echinaforce), and a novel cytokine array system designed to measure simultaneously the levels of 20 different cytokines secreted by bronchial epithelial cell cultures in response to infection. Streptococcus pyogenes (Group A Strep), which is often associated with sore throat and more severe pulmonary infections, was readily inactivated by Echinacea, which also completely reversed the cellular pro-inflammatory response. Hemophilus influenzae and Legionella pneumophila were also readily inactivated, and their pro-inflammatory responses reversed. Staphylococcus aureus (methicillin-resistant and sensitive strains) and Mycobacterium smegmatis were less sensitive to the bactericidal effects of Echinacea however, but their pro-inflammatory responses were still completely reversed. In contrast some other pathogens tested, including Candida albicans, were relatively resistant. Thus Echinaforce) exerts a dual action against several important respiratory bacteria, a killing effect and an anti-inflammatory effect. These results support the concept of using a standardized Echinacea preparation to control symptoms associated with bacterial respiratory infections.

Selection of natural health products for clinical trials: a preclinical template.

In preparation for a clinical trial on the efficacy of Echinacea products with a pediatric population, a rational method for selection of test products was developed, based on phytochemical and bioassay evaluation. Ten currently available commercial products of Echinacea angustifolia (EA) or Echinacea purpurea (EP) were selected, and 3 bottles of each of 2 different lots were purchased for each product. Investigators were blinded to product identity before phytochemical analysis. Lot-to-lot variation was small, but product variation due to species and formulation was large. Products derived from ethanol extracts had low polysaccharide content and high levels of alkamides (EA), echinacoside (EA), cynarin (EA), cichoric acid (EP), and caftaric acid (EP). These products possessed high antiviral activities that differed between EA and EP products, but limited immune activation properties. In contrast, products derived without ethanol extraction had higher polysaccharide levels, but low levels of other components. These aqueous compounds showed immunostimulant activity as measured in a mouse macrophage model and a somewhat different antiviral profile. The choice of Echinacea product for clinical trial must therefore consider the impact of immune enhancement, the specific viral infection targeted, and the potential to reduce symptoms via antiinflammatory activity. Product selection may also depend on whether the intent of the trial is prophylaxis or treatment.

Echinacea as an antiinflammatory agent: the influence of physiologically relevant parameters.

Numerous Echinacea preparations are available on the market for the prevention and treatment of cold and 'flu symptoms and inflammatory conditions associated with infections. Most of these preparations are consumed orally in the form of aqueous or ethanol extracts and tinctures. Since the recommended consumption normally involves a brief local exposure to the diluted preparation at an unspecified time in relation to the actual infection, then it is important that experimental models for the evaluation of Echinacea reflect these limitations. A line of human bronchial epithelial cells, in which rhinoviruses stimulate the production of pro-inflammatory cytokines, was used to evaluate several relevant parameters. The chemically characterized Echinacea preparation (Echinaforce) was capable of inhibiting completely the rhinovirus induced secretion of IL-6 (interleukin-6) and IL-8 (chemokine CXCL-8) in these cells, regardless of whether the Echinacea was added before or after virus infection, and in response to a range of virus doses. This inhibitory effect was also manifest under conditions resembling normal consumption with respect to the duration of exposure to Echinacea and the Echinacea dilution. It is concluded that under real life conditions of Echinacea consumption, the virus-induced stimulation of pro-inflammatory cytokines can be effectively reversed or alleviated.

Remediation of sandy soils using surfactant solutions and foams.

Remediation of sandy soils contaminated with diesel oil was investigated in bench-scale experiments. Surfactant solution, regular foams and colloidal gas aphrons were used as remediation fluids. An experimental design technique was used to investigate the effect of relevant process variables on remediation efficiency. Soils prepared with different average particle sizes (0.04-0.12 cm) and contaminated with different diesel oil contents (40-80 g/kg) were used in experiments conducted with remediation fluids. A mathematical model was proposed allowing for the determination of oil removal rate-constant (k(v)) and oil content remaining in the soil after remediation (C(of)) as well as estimation of the percentage of oil removed. Oil removal efficiencies obtained under the central experimental design conditions were 96%, 88% and 35% for aphrons, regular foams and surfactant solutions, respectively. High removal efficiencies were obtained using regular foams and aphrons, demanding small amounts of surfactant.

Modulation of immune response gene expression by echinacea extracts: results of a gene array analysis.

Echinacea extracts have traditionally been used in the treatment of many infectious and other diseases (such as rhinovirus colds), and research has revealed the presence of various bioactivities in these extracts, particularly those connected with immune responses. We examined the effects of Echinacea by using gene expression analysis in a line of human bronchial epithelial cells, with or without rhinovirus infection. More than 13 000 human genes were evaluated. From these analyses we focused primarily on immune response genes and found that both Echinacea extracts, one predominantly rich in polysaccharides and the other rich in alkylamides and caffeic acid derivatives, stimulated the expression of numerous genes. These included a number of cytokines and chemokines, although the pattern of stimulation was different. In addition, Echinacea extracts tended to neutralize the effects of the rhinovirus. When the immune response gene pathways were analyzed with the Ingenuity Pathway program, it became apparent that many of them were interconnected through a major node, the transcription factor C/EBPbeta (CAAT/enhancer-binding protein beta) and its related C/EBP proteins. This suggests that Echinacea can bring about important biological responses in cells by virtue of interactions between components of the extract and a small number of intracellular factors involved in multiple signaling pathways.

Inactivation of Norovirus by ozone gas in conditions relevant to healthcare.

We evaluated the ability of ozone gas to inactivate Norovirus and its animal surrogate feline calicivirus (FCV) in dried samples placed at various locations within a hotel room, a cruise liner cabin and an office. Norovirus was measured by quantitative reverse transcriptase real-time polymerase chain reaction (QRT-PCR) assay, and FCV by a combination of QRT-PCR and virus infectivity assays. We were able to reduce the concentration of infectious FCV by a factor of more than 10(3), and in some cases beyond detection, under optimal conditions of ozone exposure with less than an hour of total operation. QRT-PCR assays indicated similar decreases in both viral RNAs. Virus-containing samples dried onto hard surfaces (plastic, steel and glass), and soft surfaces such as fabric, cotton and carpet, were equally vulnerable to the treatment. Our results show that Norovirus can be inactivated by exposure to ozone gas from a portable commercial generator in settings such as hotel rooms, cruise ship cabins and healthcare facilities.

Echinacea extracts modulate the production of multiple transcription factors in uninfected cells and rhinovirus-infected cells.

Extracts of Echinacea purpurea are widely used for the prevention and treatment of common colds, coughs, bronchitis and other upper respiratory infections, many of which are caused by rhinoviruses (RVs). Recent reports have indicated that rhinoviruses can stimulate the release of various pro-inflammatory cytokines and chemokines from cultured nasal and bronchial human epithelial cells, and several transcription factors (TFs) have been implicated in this process. The effects of Echinacea treatment and rhinovirus infection on the activation of a range of transcription factors were evaluated by means of a protein/DNA array analysis. The BEAS-2B cell line was used as the model, and nuclear extracts of uninfected cells and rhinovirus-14 infected cells were examined with and without treatment with one of two chemically different Echinacea extracts. It was found that both Echinacea extracts increased the nuclear content of more than 30 transcription factors, including the 12 pro-inflammatory factors examined, such as NFkB, AP-1, AP-2 and STATs 1-6. Virus infection resulted in a more dramatic increase in these same TFs. However, when RV-infected cells were treated with either of the two Echinacea extracts, TF levels were reduced to low levels, although the pattern of the reductions was different for the two extracts. These results indicate that rhinovirus infection of epithelial cells, and treatment with Echinacea extracts, led to profound effects on numerous transcription factors, which could explain the previously observed modulation of secreted cytokines and chemokines, as well as other signaling pathways. In addition, the results could help to explain the beneficial effects of Echinacea consumption.

Echinacea extracts modulate the pattern of chemokine and cytokine secretion in rhinovirus-infected and uninfected epithelial cells.

Extracts of Echinacea purpurea are among the most widely used herbal medicines throughout Europe and North America for the prevention or treatment of common cold, coughs, bronchitis and other upper respiratory infections. Popular preparations include expressed juice from the aerial parts of the plant (which contain polysaccharides) and alcoholic tinctures from roots (containing caffeic acid derivatives and alkylamides). Since immune modulation has been reported for similar extracts, cytokine antibody arrays were used to investigate the changes in the pro-inflammatory cytokines and chemokines released from a cultured line of human bronchial epithelial cells exposed to Rhinovirus 14 and two different chemically characterized Echinacea extracts. Virus infection stimulated the release of at least 31 cytokine-related molecules, including several important chemokines known to attract inflammatory cells. Most of these effects were reversed by simultaneous exposure to either of the two Echinacea extracts, although the patterns of response were different for the two extracts. These results could explain the antiinflammatory properties of Echinacea extracts. Furthermore, a number of these cytokines were stimulated by the same Echinacea preparations in uninfected cells. These observations therefore provide support for the alleged beneficial uses of Echinacea extracts.

Tumor-specific and photodependent cytotoxicity of hypericin in the human LNCaP prostate tumor model.

Hypericin (HYP) has been reported to have photodependent cytotoxic activity in a variety of cancer cell lines. However, this activity has yet to be rigorously tested in vivo in tumor models. In this study LNCaP, PC-3 and DU-145 cells were used to test the cytotoxic effects of HYP in vitro, precursory to an in vivo study designed to investigate the effects of HYP in an established murine model for prostate cancer. Specifically, the model used employs immunocompromised nude mice bearing the LNCaP solid tumor xenograft. In vitro cytotoxicity experiments indicated that the dose causing 50% lethality for HYP in LNCaP, PC-3 and DU-145 cells were 2.07, 2.15 and 2.23 microM, respectively, following irradiation with red light (590 nm) for 30 min at a fluence rate of 0.1 J/cm2/s. Cells treated with HYP in the absence of photoirradiation showed no signs of cytotoxicity. A tissue distribution study was also carried out using the LNCaP solid tumor model to determine whether or not HYP is distributed to the target tissue. HYP was broadly distributed in tissues studied, including LNCaP tumor xenograft tissue. Furthermore, tumor tissue eliminated HYP at a slower rate than any of the other tissues examined. Interestingly, HYP levels were maintained in serum 24 h after oral administration (5 mg/kg dose). A pilot study designed to examine the efficacy of HYP treatment in nude mice bearing LNCaP tumors conducted over 28 days suggested that HYP, in combination with photoirradiation, inhibits both tumor growth and the elevation of prostate-specific antigen levels. Although the results reported for the current studies are preliminary they do provide evidence for an application of HYP PDT to prostate cancer which warrants further investigation.

Antiviral and antimicrobial activities of Colombian medicinal plants.

Strong antiviral and antimicrobial activities were detected in methanolic extracts of 24 plants used medicinally in the treatment of skin infections in four different regions of Colombia. Thirteen extracts displayed activity against herpes simplex virus (HSV) whereas none was active against poliovirus. The antiviral activity was indicated by a total inhibition of viral cytopathic effects (CPE) at a non-cytotoxic concentration of the extract. The most potent extract was obtained from Byrsonima verbascifolia (L.) HBK. which showed anti-HSV activity at a concentration as low as 2.5 microg/ml. Antimicrobial screening was conducted using the disc diffusion assay against Klebsiella pneumoniae, Escherichia coli, Streptococcus faecalis, Mycobacterium phlei, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium and the human pathogenic yeast, Candida albicans. Anti-Candida activity was observed for Piper lanceaefolium HBK. and Juglans neotropica Diels. Twenty-two extracts displayed activity against Gram-positive bacteria whereas none was active against the Gram-negative species. We concluded that these Colombian medicinal plants represent an untapped source of potentially useful antivirals and are worthy of further study.

Antiviral activities in plants endemic to madagascar.

In order to determine the potential of Malagasy plants as sources of antiviral activities, ethanolic extracts of 11 plants, endemic to Madagascar, were evaluated for antiviral activities. Nine of the extracts had significant activity against herpes simplex virus (HSV), whereas only four were active against Sindbis virus. Five extracts: Cynometra cloiselii , Cynometra madagascariensis , Evonymopsis longipes , Ravensara retusa , and Terminalia monoceros were particularly potent and could completely inactivate the HSV test inoculum (100 infectious virus particles) at concentrations of less than 25 µg/ml. Most of the active phytochemicals were photosensitizers. However, the combined properties of the active extracts indicated the presence of distinct compounds in different species. On the basis of these results we believe that it would be worthwhile expanding these studies to include additional species of Malagasy plants.

Investigation of medicinal plants of togo for antiviral and antimicrobial activities.

Methanol extracts were prepared from 19 medicinal plants of Togo and, by means of standard laboratory tests, were analysed for antiviral and antibiotic activities. Ten of the 19 showed significant antiviral activity and all but two displayed antibiotic activity. Extracts of three species, Adansonia digitata (the most potent), Conyza aegyptiaca and Palisota hirsuta , were active against all three test viruses (herpes simplex, Sindbis and poliovirus). The other seven, however, were more selective, showing activity against only one or two viruses. The antibiotic profiles varied considerably. The observation that each extract showed a distinctive permutation of target organisms suggests that different bioactive phytochemicals are present in each species. Only two of the extracts were devoid of bioactivity.

Further iinvestigations on the antiviral activities of medicinal plants of togo.

Further studies were done on the antiviral activities of 10 species of Togolese medicinal plants, previously shown to possess activity against herpes simplex virus (HSV). The dominant activity in all cases was virucidal (direct inactivation of virus particles), although Adansonia digitata extracts also appeared to have intracellular antiviral activities as well, which could indicate the presence of multiple antiviral compounds, or a single compound with multiple actions. In the seven most active extracts, the anti-HSV activity was considerably enhanced by light, especially UVA (long wavelength UV), although they all showed "dark" antiviral activity as well. Thus, all the extracts contained antiviral photosensitizers. In all tests, the root-bark and leaf extracts of A. digitata were the most potent.

Antiviral activities in extracts of Turkish medicinal plants.

A total of 16 ethanol extracts of Turkish medicinal plants were evaluated for antiviral activities against herpes simplex virus (HSV) and Sindbis virus (SINV). Extracts of Galanthus elwesii and Rheum ribes showed the most potent anti-HSV activities, while six other extracts had weaker activities. Galanthus elwesii and Leucojum aestivum were the most potent anti-SINV extracts with four others showing weaker activities. In total, five extracts were active against both viruses, three were selective for HSV and one was selective for SINV. Evidence for an antiviral photosensitizer was obtained in two anti-HSV extracts, in which activity was either completely dependent on light, or was con-siderably enhanced by light. Thus, several Turkish medicinal plants appear to be promising sources of antiviral activities.

Bromohypericins are potent photoactive antiviral agents.

Several hypericin derivatives, previously shown to have interesting light-mediated biological activities, were evaluated for antiviral activities against herpes simplex virus and influenza virus. Three brominated hypericins, the dibromo- and tetrabromo-derivatives and the natural compound gymnochrome B were all very active against both viruses, particularly herpes simplex virus, although light was required in all cases for maximum activity. The dibromohypericin was the most potent, under standard assay conditions, gymnochrome B was approximately as active as hypericin itself and tetrabromohypericin significantly less so. Surprisingly, hexamethylhypericin, which is known to have potent anti-protein kinase (PK) C activity, as well as anticell proliferation properties, showed no antiviral activity at all. The compounds were also evaluated in different serum concentrations. All the active compounds were inhibited by increasing concentrations of serum, but to different degrees, such that their relative antiviral potencies changed to some extent. Thus, in summary, there was no correlation between antiviral and anti-PK or anticellular activities, and consequently it is not possible at present to define those structural features of hypericin-type molecules that are required for their various biological activities.

Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm.

Dorsal mesoderm induction in arthropods and ventral mesoderm induction in vertebrates are closely related processes that involve signals of the BMP family. In Drosophila, induction of visceral mesoderm, dorsal muscles, and the heart by Dpp is, at least in part, effected through the transcriptional activation and function of the homeobox gene tinman in dorsal mesodermal cells during early embryogenesis. Here we present a functional dissection of a tinman enhancer that mediates the Dpp response. We provide evidence that mesoderm-specific induction of tinman requires the binding of both activators and repressors. Screens for binding factors yielded Tinman itself and the Smad4 homolog Medea. We show that the binding and synergistic activities of Smad and Tinman proteins are critical for mesodermal tinman induction, whereas repressor binding sites prevent induction in the dorsal ectoderm and amnioserosa. Thus, integration of positive and negative regulators on enhancers of target genes appears to be an important mechanism in tissue-specific induction by TGF-beta molecules.