A sensitive LC-MS/MS method for the quantitative analysis of the Echinacea purpurea constituent undeca-2-ene-8,10-diynoic acid isobutylamide in human plasma

Echinacea purpurea is one of the most popular herbal medicines and is known for its immunostimulatory effects. Alkylamides are the main lipophilic components of E. purpurea that contribute to its pharmacological actions. For quantification in human plasma of one of these alkylamides, undeca-2-ene-8,10-diynoic acid isobutylamide, a sensitive LC-MS/MS assay has been developed and validated. Plasma samples were pretreated using liquid-liquid extraction with a mixture of diethyl ether and n-hexane (50:50, v/v). Dried extracts were reconstituted in 50 μL of acetonitrile-water (50:50, v/v) after which 15 μL of sample was injected into the HPLC system. HPLC was performed using a Polaris 3 C18-A column (50 mm×2 mm ID) and isocratic elution with acetonitrile-water (50:50, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. Subsequently, electrospray ionization in the positive ion mode followed by tandem mass spectrometry was performed for detection. The total run time was 3 min. The assay was validated over a concentration range from 0.05 to 50 ng/mL for undeca-2-ene-8,10-diynoic acid isobutylamide, with 0.05 ng/mL being the lower limit of quantification using 1.0 mL plasma samples. Inter-assay inaccuracy (±12.7%), within-day and between-day precisions (CV≤8.23%) were acceptable. Further, undeca-2-ene-8,10-diynoic acid isobutylamide was found to be chemically stable under relevant conditions. Finally, the applicability of this assay has been successfully demonstrated in a pharmacokinetic experiment in which a human volunteer ingested a commercial extract of E. purpurea.     

Phenetic comparison of sevenEchinacea species based on immunomodulatory characteristics

The purpose of the present investigation was to compare similarities and differences in immune response among Echinacea species, which are commonly used to treat upper respiratory infections. The investigation involved two components: acquisition of immunomodulatory data reported here for the first time, and combined phenetic analysis of these data along with previous reports. Experimental data were obtained by stimulating human PBMC in vitro with extracts from Echinacea spp. and assaying production of three cytokines (interleukin-1β [IL-1β] interleukin-2 [IL-2], and tumor necrosis factor α [TNF-α]). Phenetic analyses were employed to compare responses across the entire data set, including UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and neighbor-joining methods. In the immune experiments conducted for this investigation, E. angustifolia,E. paradoxa, E. purpurea, E. simulata, andE. tennesseensis extracts significantly augmented IL-1 β and TNF-α production, whereas no extracts significantly modulated IL-2. All phenetic methods produced similar dendrograms, revealing two species pairs (E. angustifolia + E. simulata and E. pallida + E.sanguinea) where both species cluster tightly and have similar immune-response profiles. These two species-pairs are maximally dissimilar from each other. The remaining species (E. paradoxa, E. purpurea, and E. tennesseensis) occupy intermediate positions in the dendrogram. Our results suggest that Echinacea spp. act heterogeneously on immune function. The utility of these data for science and industry is discussed.     

Cotinus coggygria Scop.: An overview of its chemical constituents,pharmacological and toxicological potential

The Anacardiaceae Lindl. family comprises of many species which are used in nutrition and in traditional folk medicine for the treatment of several human diseases. Cotinus coggygria Scop. commonly known as “smoke tree”, is a commercial ornamental plant with high medicinal usages, belongs to the family Anacardiaceae. The present review provides a comprehensive report of empirical investigations on important pharmacological activities and phytochemical screening of essential oils and extracts. Relevant information was collected from scientific journals, books, and reports via library and electronic search using Medline, PubMed, Google Scholar, ScienceDirect, Web of Science, and Scopus. The plant has been extensively investigated in a broad range of studies to provide scientific evidence for folklore claims or to find new therapeutic uses. Numerous activities namely antioxidative, antibacterial, antifungal, antiviral, anticancer, antigenotoxic, hepatoprotective and anti-inflammatory have been demonstrated for all parts of these plants by in vivo and in vitro studies. Essential oils and extracts showed various pharmacological and biological properties which make them an effective remedy for various kinds of illnesses. Considering data from the literature, it could be demonstrated that C. coggygria possesses diverse bioactive properties and immense utilization in medicine, health care, cosmetics and as health supplements.     

Morphology and anatomy of <Emphasis Type="Italic">Echinacea purpurea,E. angustifolia,E. pallida</Emphasis> and <Emphasis Type="Italic">Parthenium integrifolium</Emphasis>

The Echinacea species are native to the Atlantic drainage area of the United States of America and Canada. They have been introduced as cultivated medicinal plants in Europe. Echinacea purpurea, E. angustifolia and E. pallida are the species most often used medicinally due to their immune-stimulating properties. This review is focused on morphological and anatomical characteristics of E. purpurea, E. angustifolia, E. pallida, because various species are often misidentified and specimens are often confused in the medicinal plant market.     

Are the health attributes of lycopene related to its antioxidant function?

A variety of epidemiological trials have suggested that higher intake of lycopene-containing foods (primarily tomato products) or blood lycopene concentrations are associated with decreased cardiovascular disease and prostate cancer risk. Of the carotenoids tested, lycopene has been demonstrated to be the most potent in vitro antioxidant leading many researchers to conclude that the antioxidant properties of lycopene are responsible for disease prevention. In our review of human and animal trials with lycopene, or lycopene-containing extracts, there is limited support for the in vivo antioxidant function for lycopene. Moreover, tissue levels of lycopene appear to be too low to play a meaningful antioxidant role. We conclude that there is an overall shortage of supportive evidence for the “antioxidant hypothesis” as lycopene’s major in vivo mechanism of action. Our laboratory has postulated that metabolic products of lycopene, the lycopenoids, may be responsible for some of lycopene’s reported bioactivity.     

Effect on prolactin secretion of Echinacea purpurea, Hypericum perforatum and Eleutherococcus senticosus

It has been recently reported that prolactin (PRL) plays an important role in immune system regulation. In this study we investigated the activity of three natural drugs with immunomodulatory activity: Echinacea purpurea (EP), Hypericum perforatum (HP) and Eleutherococcus senticosus (ES) on PRL production. Male rats were orally treated with two different doses (30 and 100 mg/kg) of extract of these drugs for 3 or 15 days. A 3-day treatment was not able to modify PRL serum levels, whereas a 15-day treatment with EP and HP at the higher dose significantly inhibits PRL production. A treatment with ES was always ineffective. A possible mechanism for this effect could be that both HP and EP extracts display a direct dopaminergic activity, although an involvement of the GABA-ergic system cannot be excluded.     

Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC

A fast and reliable HPLC method for the determination of caffeic acid derivatives (caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid) in various species of the genus Echinacea has been developed. Extraction of root samples by magnetic stirring with 80% methanol aqueous solution at room temperature allowed the complete recovery of all compounds of interest. Root extracts were analysed on a reversed-phase column with gradient elution and photodiode array detection. Caffeic acid derivatives showed differential qualitative and quantitative distributions in Echinacea species. The total amount of phenolic compounds ranged from 33.95 to 0.32 mg/g. The highest contents of caffeic acid derivatives were found in E. paradoxa var. paradoxa, E. paradoxa var. neglecta and E. purpurea, followed by E. angustifolia var. angustifolia, E. simulata, E. pallida and E. laevigata, whilst E. tennesseensis, E. sanguinea and E. atrorubens had low amounts of phenolic compounds. The radical scavenging activities of methanolic extracts of roots of Echinacea species was evaluated in vitro using the DPPH* radical scavenging method. The EC50 values of the samples ranged from 122 to 1223 microg/mL. The radical scavenging activities of the root extracts were correlated with the content of phenolic compounds, with a correlation coefficient (r2) of 0.923.     

Escherichia coli reactions to the action of the cationic proteins of blood leukocytes

The antibacterial effect of cation proteins (CP) of swine leukocytes with respect to Escherichia coli strain 17 has been demonstrated in vitro. The composition and properties of these CP have been studied; as a result, a comparatively high content of basic and dicarbonic amino acids and the presence of protease activity have been established. The release of the components of the metabolic fund by E. coli under the influence of CP of swine leukocytes is indicative of changes in the permeability of microbial membranes and, probably, of the deficiency of energy resources. The addition of heparin to CP of swine leukocytes levels their antibacterial effect with respect to E. coli.     

The pharmacognosy of Humulus lupulus L. (hops) with an emphasis on estrogenic properties

As the population ages, there is an ever-increasing need for therapeutic agents that can be used safely and efficaciously to manage symptoms related to postmenopausal estrogen deficiency. Endogenous estrogens, e.g., 17beta-estradiol, of exogenous mammalian origin, e.g., horses, have long been used to manage such symptoms. There are more than 20 different classes of phytochemicals that have demonstrated affinity for human estrogen receptors in vitro. Some studies on exogenous estrogenic substances of botanical origin (phytoestrogens), such as standardized formulations of plant extracts with in vitro and in vivo estrogenic activity from soy (Glycine max Merill.) and red clover (Trifolium pratense L.), suggest clinical efficacy. Few clinical data for phytoestrogens other than isoflavonoids are available. In an exhaustive review of the literature through 2003, only two clinical trials were identified that were designed to evaluate the effect of hops (Humulus lupulus L.) on symptoms related to menopause. Folkloric, chemical, and biological literature relating primarily to the use of hops for their estrogenic activity, and two human clinical trials, are reviewed.     

Influence of Echinacea purpurea intake during pregnancy on fetal growth and tissue angiogenic activity

The process of angiogenesis and control of blood vessels sprouting are fundamental to human health, as they play key roles in many physiological and pathological conditions. Intake of different pharmaceuticals with antiangiogenic activity by pregnant women may lead to severe developmental disturbances as it was described in case of thalidomide. It may also cause immunomodulatory effects as it was shown for antibiotics, theobromine, caffeic acid or catechins on the pregnant mice model. At present, Echinacea purpurea-based phytoceuticals are among the most popular herbals in the marketplace. Many compounds of Echinacea extracts (polysaccharides, alkamides, polyphenols, glycoproteins) exert immunomodulatory, anti-oxidative and anti-inflammatory activity. Echinacea is one of the most powerful and effective remedies against many kinds of bacterial and viral infections. In previous studies we shown significant inhibitory effect of the Echinacea purpurea based remedy on tumour angiogenic activity using cutaneous angiogenesis test, and an inhibitory effect on L-1 sarcoma growth was observed . The aim of the present study was to establish whether pharmaceuticals containing alcoholic extracts of Echinacea purpurea given to pregnant mice influence angiogenic activity and tissue VEGF and bFGF production of their fetuses. We showed that angiogenic activity of tissue homogenates was increased in Esberitox group and diminished in case of Immunal forte as compared to standard diet group. In case of Echinapur group we did not find significant differences in angiogenic activity. VEGF and bFGF concentration were lower in all groups compared to the control. In the case of Echinapur and Esberitox number of fetuses in one litter were slightly lower as compared to control group, but the difference is on the border of statistical significance. In conclusion, there is some possibility that pharmaceuticals containing Echinacea purpurea might influence fetal development in human also, because they may interfere with embrional angiogenesis , and should not be recommended for pregnant women.     

A reappraisal of leukocyte dehydroascorbate reductase

Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was determined in human leukocyte homogenates using a direct spectrophotometric assay. Despite previous studies, using a less sensitive coupled assay, which reported that this enzyme was present in leukocytes, we found that neither neutrophil nor chronic lymphocytic leukemia lymphocyte extracts had detectable activity. Furthermore, when the product was quantitated by HPLC, protein-dependent generation could not be demonstrated. Mixing experiments with a partially purified enzyme preparation from spinach leaves provided no evidence for the presence of an inhibitor in neutrophil homogenates. These findings suggest that in human leukocytes, dehydroascorbate reduction does not occur enzymatically.     

In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes

Lentinula edodes, known as “shiitake” is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC₅₀ values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI_comit/SI_mit ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.     

Steroid sulfatase activities in human leukocytes: biochemical and clinical aspects.

Steroid sulfatase is a membrane-bound microsomal enzyme, present in various tissues. In this report, data on sulfatase activity in peripheral blood leukocytes isolated from normal women and the characterization of its enzyme are studied. In addition, sulfatase activities in placental sulfatase deficiency (PSD) and ichthyosis patients including ichthyosis vulgaris (IV) and recessive X-linked ichthyosis (RXLI) were analysed and were compared with normal subjects. Steroid sulfatase activity was measured by using tritium labeled steroid sulfate as the reaction substrate. It is demonstrated that human leukocytes contain a sulfatase activity for pregnenolone sulfate (P5-S), dehydroepiandrosterone sulfate (DHA-S) and estrone sulfate (E1-S) respectively. This enzyme has a greatest affinity for P5-S, but the activity for E1-S was the highest among the three substrates. The steroid sulfatase activity in female leukocytes is significantly stronger than that in normal males (p less than 0.001) as determined by the cleavage of DHA-S. Sulfatase in leukocytes obtained from the PSD babies and RXLI patients had lower sensitivity. In the case of the mother affected with PSD, the activity was less than half of that in normal men (p less than 0.001) and the levels did not overlap with that in normal women. In patients with IV, the activities were in the normal ranges for both males and females. The measurement of leukocyte sulfatase activity would be a clinically useful tool for the diagnosis of PSD carriers and pedigree analysis.     

Expression of human alpha-l-fucosyltransferase gene homologs in monkey kidney COS cells and modification of potential fucosyltransferase acceptor substrates by an endogenous glycosidase

Previous investigations on the monkey kidney COS cell line demonstrated the weak expression of fucosylated cell surface antigens and presence of endogenous fucosyltransferase activities in cell extracts. RT-PCR analyses have now revealed expression of five homologs of human fucosyltransferase genes, FUT1, FUT4, FUT5, FUT7, and FUT8, in COS cell mRNA. The enzyme in COS cell extracts acting on unsialylated Type 2 structures is closely similar in its properties to the alpha1,3- fucosyltransferase encoded by human FUT4 gene and does not resemble the product of the FUT5 gene. Although FUT1 is expressed in the COS cell mRNA, it has not been possible to demonstrate alpha1,2- fucosyltransferase activity in cell extracts but the presence of Le(y) and blood-group A antigenic determinants on the cell surface imply the formation of H-precursor structures at some stage. The most strongly expressed fucosyltransferase in the COS cells is the alpha1,6-enzyme transferring fucose to the innermost N -acetylglucosamine unit in N - glycan chains; this enzyme is similar in its properties to the product of the human FUT8 gene. The enzymes resembling the human FUT4 and FUT8 gene products both had pH optima of 7.0 and were resistant to 10 mM NEM. The incorporation of fucose into asialo-fetuin was optimal at 5.5 and was inhibited by 10 mM NEM. This result initially suggested the presence of a third fucosyltransferase expressed in the COS cells but we have now shown that triantennary N- glycans with terminal nonreducing galactose units, similar to those present in asialo-fetuin, are modified by a weak endogenous beta-galactosidase in the COS cell extracts and thereby rendered suitable substrates for the alpha1,6- fucosyltransferase.      

Variations in surface protein composition associated with virulence properties in opacity types of Neisseria gonorrhoeae.

The biological properties of a series of opacity variants of Neisseria gonorrhoeae P9 have been examined. A novel protein, designated protein IId* (mol. wt 28 850), was identified within the set of heat-modifiable surface proteins previously reported. All variants producing extra outer membrane proteins were less sensitive to the bactericidal action of serum than the prototype transparent strain, with protein IIa* (mol. wt 28 500) being associated with increased resistance. The production of a different protein, protein II* (mol. wt 29 000), was correlated with resistance to low molecular weight antimicrobial agents (penicillin, fusidic acid, Cu2+, Zn2+). Increased adhesion to human buccal epithelial cells was demonstrated in all variants that produced extra surface proteins. These variants did not show increased binding to hexyl- and phenyl-substituted Sepharose gels suggesting that hydrophobic interaction was not responsible for their cohesive properties. The prototype strain lacking additional proteins demonstrated the greatest binding to erythrocytes, indicating that adhesion to buccal cells and red blood cells is mediated by different mechanisms. One variant producing protein IIa* showed increased association with leukocytes, whereas another producing protein IIb* showed decreased association with leukocytes. These results show that the heat-modifiable surface proteins are important virulence attributes of the gonococcus: this must be considered in the selection of strains for vaccine trials.     

Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals

With the use of the spin trapping methods, the scavenging effects of the extracts of green tea and other natural foods are studied. In stimulated polymorphonuclear leukocytes (PMN) system, water extract fraction 6 (F6) from green tea and green tea polyphenols (GTP) have the strongest scavenging effect on the active oxygen radicals, much stronger than vitamin C (Vc) and vitamin E (VE). Rosemary antioxidants (RA) and Curcumin (Cur) have weaker scavenging effects than Vc, but stronger than VE. In Fenton Reaction, Cur has the strongest scavenging effect (69%) on hydroxyl radicals. In irradiation, riboflavin system F6(74%) and GTP(72%) have very strong scavenging effects that are weaker than Vc, but much stronger than VE (23%). With the use of spin probe oxymetry, the oxygen consumption in respiratory burst of stimulated PMN were measured when the antioxidants existed in these systems. The results demonstrated that these antioxidants did not affect the respiratory burst of human polymorphonuclear leukocytes stimulated with PMA.     

Is the Western diet adequate in copper?

Copper has been known to be essential for health for more than three quarters of a century. Myriad experiments with animals reveal that the cardiovascular, musculoskeletal and nervous systems are most sensitive to deficiency. Copper in the Western diet has been decreasing at least since the 1930s; half of the adult population consumes less than the amount recommended in the European Communities and the United Kingdom. At least one fourth of adults consume less than the estimated average requirement published for the United States and Canada. Hundreds of people have been reported in journals about medicine and neurology rather than nutrition to have impaired copper nutriture based on the criteria of low copper concentrations and low activities of enzymes dependent on copper in various fluids and tissues. In contrast, only 46 people have participated in depletion/repletion experiments needed to define requirements. Almost 1000 people have benefited from supplements containing copper in controlled trials. People deficient in copper are being identified increasingly; it is unknown if unusually high requirements or unusually low diets are causal. Alzheimer's disease, ischemic heart disease and osteoporosis are the most likely human illnesses from low copper intakes.     

Peptidase activities of the 20/26S proteasome and a novel protease in human brain

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.     

Steroid 5alpha-reductase in cultured human fibroblasts. Biochemical and genetic evidence for two distinct enzyme activities.

Various properties of the steroid 5alpha-reductase have been examined in cell-free extracts of skin and of fibroblasts cultured from genital and nongenital skin from control subjects and from patients with several forms of male pseudohermaphroditism. When 20alpha-hydroxy-4-[1,2-3H] pregnen-3-one was used as substrate, two 5alpha-reductase activities could be demonstrated in intact skin and cultured fibroblasts. The major activity, previously described for microsomes from human prepuce and extracts of cultured foreskin fibroblasts, is characterized by a narrow pH optimum near 5.5 and is limited to fibroblasts derived from genital skin. A second activity, not limited by the site of biopsy, has been demonstrated over a higher and broader range of pH (from 7 to 9); this enzyme activity is found in both genital and nongenital skin and in fibroblasts cultured from all skin regions. Whereas there is wide variability in the activity at pH 5.5 in genital skin fibroblasts, the activity at pH 7 to 9 is similar in fibroblasts derived from all anatomical sites. The two activities exhibit different kinetics with respect to steroid substrate and are also dissimilar in their subcellular distributions. Other properties, such as coenzyme requirement, steroid substrate specificity, and instability with increasing temperature, appear to be similar.