Anti-Respiratory Syncytial Virus Compounds from Two Endophytic Fungi Isolated from Nigerian Medicinal Plants.

Background: Respiratory syncytial virus (RSV) is known to cause severe respiratory infections particularly in infants younger than 2 years of age. The only approved drug, ribavirin, is expensive and is not likely to improve therapeutic outcome, thereby necessitating the search for safer and more potent alternatives from natural sources such as endophytic fungi. The present study aimed to investigate the anti-RSV activity of compounds from endophytic fungi. Methods: Two endophytic fungi Colletotrichum gloeosporioides and Pestalotiopsis thea were isolated from the fresh leaves of the host Nigerian plants Anthocleista djalonensis and Fagara zanthoxyloides, respectively. After fermentation in solid rice media, C. gloeosporioides afforded 4 known compounds 4-hydroxybenzoic acid (1), vanillic acid (2), ferulic acid (3) and Nb-acetyltryptamine (4) while P. thea afforded 3 known compounds chloroisosulochrin (5), ficipyrone A (6) and pestheic acid (7). The compounds were investigated for their anti-RSV activity using the HEP-2 cell lines and ribavirin as the standard drug. Results: Compound 5 was found to show the strongest inhibition of the RSV with IC50 of 4.22±1.03 µM (ribavirin 4.91±1.85 µM). Other compounds showed moderate inhibition of the virus (IC50 ranging from 45.00±0.98 to 259.23±2.36 µM). Conclusion: The results of the present study have shown that chloroisosulochrin (5), isolated from an endophytic fungus P. thea, possesses strong activity against RSV.

Fungal endophytes - secret producers of bioactive plant metabolites.

The potential of endophytic fungi as promising sources of bioactive natural products continues to attract broad attention. Endophytic fungi are defined as fungi that live asymptomatically within the tissues of higher plants. This overview will highlight the uniqueness of endophytic fungi as alternative sources of pharmaceutically valuable compounds originally isolated from higher plants, e.g. paclitaxel, camptothecin and podophyllotoxin. In addition, it will shed light on the fungal biosynthesis of plant associated metabolites as well as new approaches developed to improve the production of commercially important plant derived compounds with the involvement of endophytic fungi.

The effects of Phyllanthus niruri aqueous extract on the activation of murine lymphocytes and bone marrow-derived macrophages.

Phyllanthus niruri L. (Euphorbiaceae) is acclaimed world-wide for its versatile ethnomedicinal uses. It features in recipes used by some herbalists to manage different diseases, including claims of efficacy against many life-threatening infections, such as HIV/AIDS and hepatitis. In order to understand the mechanisms and the involvement of the immune system in mediating these activities, the effects of the aqueous extract of P. niruri on the activation of murine lymphocytes and macrophages were investigated. The study showed that the extract of P. niruri is a potent murine lymphocytes mitogen, inducing significant (p < 0.01) increases in the expression of surface activation maker (CD69) and proliferation of B and T lymphocytes. The production of interferon-gamma (IFN- gamma) and interleukine-4 (IL-4) by P. niruri extract-stimulated naïve splenocytes cultures was also significantly (p < 0.05) increased in a concentration-dependent manner. Various indices of activation and functions murine bone marrow-derived macrophages were significantly (p < 0.05) enhanced by pre-treatment with the extract, including phagocytosis, lysosomal enzymes activity, and TNF-alpha release. Phyllanthus niruri extract was also shown to modulate nitric oxide release by macrophages. These activities suggest that stimulation of the immune system by the extracts of P. niruri could be partly responsible for the ethnomedicinal applications in the management of infectious diseases.

Adjuvant properties of AcF1, an immunostimulant fraction of Alchornea cordifolia extract.

As a result of strong experimental data supporting effectiveness and safety, herb-based immunomodulators are paving way as alternative sources of potent adjuvants for vaccines. In this study, the immunostimulatory and adjuvant properties of AcF1, a flavonoids-rich fraction of Alchornea cordifolia extract, was evaluated. In vitro, AcF1 was shown to activate total splenocytes, CD4+ T cells, and B cells, inducing remarkable increases in CD69 expression, profound proliferation, and increased IL-4 and IFN-gamma expression by the naïve splenic cells in a concentration-dependent manner. Lympho-activation and proliferation induced by AcF1 was partially inhibited by U0126, a selective mitogen activated protein kinase kinase (MKK) inhibitor. Additionally, AcF1 was shown to induce structural and functional maturation of bone marrow-derived dendritic cells (BM-DCs) and their specific-antigen presentation functions. Used as an adjuvant in a homologous prime-boost OVA immunisation in C57BL/6 mice, AcF1 significantly (P<0.05) increased the level of OVA-specific antibody titres in the sera of immunised mice, compared to the control group immunised with OVA alone. The results of this study show AcF1 as a potent immunostimulant and a potential adjuvant for further study in combination with other vaccine antigens.

Effects of cycloartane saponins from hairy roots of Astragalus membranaceus Bge., on human tumor cell targets.

For the first time three different natural compounds, isolated from hairy roots of Astragalus membranaceus, cultivated in airlift bioreactor were tested for their cytotoxic potential and apoptosis induction in a panel of human tumor cell lines. Root cultures, cultivated in bioreactor gave 18.5 g l(-1) dry wt roots with the highest astragaloside production in vitro up to now - 1.64% (astragaloside I), 1.12% (astragaloside II) and 1.08% (astragaloside III). In this manner the production in airlift bioreactor can be used as means of reliable supply of cycloartane saponins to extend the research to human clinical studies.

Rocaglamide sensitizes leukemic T cells to activation-induced cell death by differential regulation of CD95L and c-FLIP expression.

Drugs with tumor selectivity may have an important benefit in chemotherapies. We have previously shown that Rocaglamide(s), derived from the medicinal plant Aglaia, kills various leukemic cells through the mitochondrial apoptosis pathway with only minor toxicities to normal lymphocytes. Here, we show further that Rocaglamide preferentially promotes activation-induced cell death in malignant T cells by differential regulation of c-FLIP and CD95L expression. Rocaglamide enhances and also prolongs activation-induced JNK activation in malignant T cells leading to downregulation of c-FLIP but upregulation of CD95L expression. We also show that malignant T cells express a significantly higher amount of Bid - the molecular linker that bridges the receptor-mediated to the mitochondria-mediated apoptosis pathway. Conversely, a substantially lower amount of c-FLIP in response to T-cell stimulation compared to normal T cells is observed. This difference may provide a therapeutic window for cancer treatment. The effect of Rocaglamide on sensitization of activation-induced cell death in malignant T cells was further demonstrated in vivo in a mouse model. Our study demonstrates that Rocaglamide may be a potential anticancer drug that simultaneously targets both c-FLIP and CD95L expressions in tumor cells. This study may also provide a new clue to design a more efficient chemotherapy by using a combination of stimuli that engage the receptor-mediated and the mitochondria-mediated death pathway.

Hypoglycaemic constituents of Stachytarpheta cayennensis leaf.

The aqueous infusion (tea) of Stachytarpheta cayennensis leaves is used ethnomedically in Peru, Nigeria and other tropical countries for the management of diabetes. Oral administration (p. o.) of aqueous (125 mg/kg) and methanolic (2000 mg/kg) extracts of the leaves to alloxan-diabetic rats showed significant blood glucose reductions by 43 and 53%, respectively, at the end of a 4 hour period similar to the strong effect of glibenclamide (5 mg/kg, P. O.). The methanolic extract was successively partitioned into ethyl acetate, butanol and water fractions, and the same test showed that the butanol fraction (2000 mg/kg) had the highest (50%) hypoglycaemic activity at 4 hours after oral administration. It was also the most active fraction when tested in vitro [insulin release from an insulin secreting cell line (INS-1)] and was also active in normal rats and rats made hyperglycaemic by a glucose load. Its activity was comparable to that of glibenclamide (positive control) in these models. This active butanol fraction was subjected to chromatographic subfractionation; some subfractions reduced hyperglycaemia in alloxan-diabetic rats to 60 and 78% and induced insulin release from the INS-1 cells; other subfractions, however, gave hyperglycaemic activities IN VIVO and inhibition of insulin release from the INS-1 cells. Three major compounds of the butanol fraction were isolated and characterised as 6beta-hydroxyipolamide, ipolamide and isoverbascoside; they increased insulin secretion from INS-1 cells to 125, 128 and 127%, respectively, whereas glibenclamide increased insulin secretion to 157%. The results justify the ethnomedical use of the plant in the management of diabetes and suggest that the butanol fraction and some of its isolated constituents mediate their actions primarily by stimulating insulin release directly.

Two new 18-en-oleane derivatives from marine mangrove plant, Barringtonia racemosa.

Two new triterpenoids, olean-18-en-3beta-O-E-coumaroyl ester (1) and olean-18-en-3beta-O-Z-coumaroyl ester (2), were isolated from the stem bark of marine mangrove plant Barringtonia racemosa, along with five known compounds, germanicol, germanicone, betulinic acid, lupeol, and taraxerol. Their structures were determined mainly by spectroscopic methods.

In vitro pharmacodynamic evaluation of antiviral medicinal plants using a vector-based assay technique.

AIMS: Medicinal plants are increasingly being projected as suitable alternative sources of antiviral agents. The development of a suitable in vitro pharmacodynamic screening technique could contribute to rapid identification of potential bioactive plants and also to the standardization and/or pharmacokinetic-pharmacodynamic profiling of the bioactive components. METHODS AND RESULTS: Recombinant viral vectors (lentiviral, retroviral and adenoviral) transferring the firefly luciferase gene were constructed and the inhibition of viral vector infectivity by various concentrations of plant extracts was evaluated in HeLa or Hep2 cells by measuring the changes in luciferase activity. Cytotoxicity of the extracts was evaluated in parallel on HeLa or Hep2 cells stably expressing luciferase. Amongst the 15 extracts screened, only the methanol (ME) and the ethyl acetate (ET) fractions of the lichen, Ramalina farinacea specifically reduced lentiviral and adenoviral infectivity in a dose-dependent manner. Further, chromatographic fractionation of ET into four fractions (ET1-ET4) revealed only ET4 to be selectively antiviral with an IC50 in the 20 microg ml(-1) range. Preliminary mechanistic studies based on the addition of the extracts at different time points in the viral infection cycle (kinetic studies) revealed that the inhibitory activity was highest if extract and vectors were preincubated prior to infection, suggesting that early steps in the lentiviral or adenoviral replication cycle could be the major target of ET4. Inhibition of wild-type HIV-1 was also observed at a 10-fold lower concentration of the extract. CONCLUSIONS: The vector-based assay is a suitable in vitro pharmacodynamic evaluation technique for antiviral medicinal plants. The technique has successfully demonstrated the presence of antiviral principles in R. farinacea. SIGNIFICANCE AND IMPACT OF STUDY: Potential anti-HIV medicinal plants could rapidly be evaluated with the reported vector-based technique. The lichen, R. farinacea could represent a lead source of antiviral substances and is thus worthy of further studies.

Rocaglamides, glycosides, and putrescine bisamides from Aglaia dasyclada.

A phytochemical analysis of the leaves of Aglaia dasyclada collected in Yunnan Province (People's Republic of China) yielded five cyclopentabenzofurans (1-5) of the rocaglamide family that are common secondary metabolites of Aglaia species as well as four biogenetically related compounds of the aglain (7), aglaforbesin (8) and forbaglin (9, 10) types. In addition, the cinnamic acid amide dasyclamide (6), which is a putative biogenetic precursor of these compounds (7-10), was isolated. The structures of the new compounds (6-10) were assigned unambiguously from the combined use of 1D and 2D NMR spectroscopy and mass spectrometry.

Aglacins A-D, first representatives of a new class of aryltetralin cyclic ether lignans from Aglaia cordata.

Four new metabolites, aglacins A-D (1-4), were identified from the methanol extract of the stem bark of Aglaia cordata. These compounds represent a new class of aryltetralin cyclic ether lignan. The structure of aglacin A (1) including the absolute configuration was elucidated by interpretation of spectral data, X-ray crystal structure determination, and employing the modified Mosher's method. In addition, three other derivatives, aglacins B-D (2-4), were isolated and identified by spectral means.

Qualitative and Quantitative Analysis of the Phenolic Constituents from Orthosiphon aristatus.

ORTHOSIPHON ARISTATUS (Orthosiphonis folium DAB 9) was studied with regard to its phenolic constituents. Twenty compounds were isolated and identified on the basis of their spectral characteristics. The compounds included nine lipophilic flavones, two flavonol glycosides, and nine caffeic acid derivatives. The presence of the recently reported methylripariochromene A could not be confirmed. All compounds identified were quantified by HPLC. The caffeic acid derivatives including the major compounds rosmarinic acid and 2,3-dicaffeoyltartaric acid (67% of total identified phenolics) predominated over the flavones (33%) in an aqueous MeOH extract. The predominance of the caffeic acid derivatives was even more pronounced in a hot water extract (94.5% of total identified phenolics) that was comparable to a herbal tea.

Accumulation and Biotransformation of Chromenes and Benzofurans in a Cell Suspension Culture of Ageratina adenophora.

A cell suspension culture of AGERATINA ADENOPHORA was shown to yield several novel chromene and benzofuran derivatives in minute amounts that were different to the compounds found in seedlings of the same species. The structure elucidation of the new compounds is described. When two of the seedling chromenes (demethoxyencecalin and demethylencecalin) were fed to the cell suspension culture, one biotransformation product each was obtained in high yields (80%) that originated from a hydroxylation at one of the geminal methyl groups of the chromene heterocycle. These products accumulated largely in the growth media even though the presence of cells was necessary for the biotransformations to occur. When the third seedling chromene (encecalin) was fed to the cell auspension culture, no significant biotransformation was noted but several of the benzofurans present as cell culture metabolites showed a significantly increased accumulation in the growth media of the treated cultures. This increased accumulation of benzofurans was found to be inducible also by adding yeast extract to the cell culture. The metabolism of chromenes and bezofurans in the cell suspension culture is discussed.

New dihydroflavonols from enceliopsis and geraea.

Phytochemical analysis of leaves from ENCELIOPSIS NUDICAULIS, E. NUDICAULIS var. CORRUGATA, and GERAEA CANESCENS afforded numerous methylated flavonoids present as resin constituents as well as benzofurans and related compounds extracted from roots. Five new dihydroflavonols present on the leaf surfaces were identified based on their spectral data and on those of the corresponding flavonols obtained by oxidation with NaHSO (3).

Prenylated Phenolics that Cause Contact Dermatitis from Glandular Trichomes of Turricula parryi.

The principal phenolic constituents were isolated from the exudate of the glandular trichomes of TURRICULA PARRYI, a California shrub that causes severe contact dermatitis. The phenolic constituents consist of a complex mixture of methoxylated flavones and derivatives of both farnesylhydroquinone and 3-farnesyl- P-hydroxybenzoic acid in which the C-7 methyl of farnesyl is replaced by hydroxymethyl, formyl and carboxyl groups. Elicitors of allergic contact dermatitis were identified with guinea pigs sensitized to the crude exudate. The principal agents of dermatitis were the farnesyihydroquinone derivatives 3 and 6.