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.

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.

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.

Broad spectrum antiviral fractions from the lichen Ramalina farinacea (L.) Ach.

BACKGROUND: An ethylacetate-soluble fraction (ET4) from the lichen Ramalina farinacea has previously been shown to inhibit the infectivity of lentiviral and adenoviral vectors, as well as wild-type HIV-1. We now determined the antiviral activity of ET4 against other wild-type viruses, including the herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV). METHODS: Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of ET4 for 30 min at 37 degrees C before adding to P4CCR5 indicator cell line (HIV-1), ELVIS TM indicator cell line (HSV-1) or HEp2 cell line (RSV) in 96-well microtitre plates. Controls contain virus alone without ET4. The anti-HIV and anti-HSV activities were quantified by estimating beta-galactosidase expression of the respective indicator cell lines while the anti-RSV activity was determined via an immunofluorescent technique, employing monoclonal mouse antibody against the P-protein of RSV. Toxicity of ET4 to cell lines was evaluated in parallel using either the BrdU incorporation method or the MTT method. The effect of ET4 on the enzymatic activity of HIV-1 reverse transcriptase was also evaluated using a chemiluminescent reverse transcriptase assay. Bioassay-guided fractionation of the whole methanol extract of R. farinacea involved sequential screening of HPLC fractions using a vector-based assay technique. RESULTS: ET4 inhibited HSV-1 and RSV potently (IC(50)=6.09 and 3.65 microg/ml, respectively). Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle and the entry step of the RSV replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 reverse transcriptase with an IC(50) of 0.022 microg/ml. Bioassay-guided fractionation of ET4 led to the identification sub-fraction rfO, with activity against lentiviral vector and HIV-1 (RNA viruses) but not against HSV-1 (DNA virus) and sub-fraction rfM, with activity against HSV-1 but not against the lentiviral vector. CONCLUSIONS: ET4 represents a novel fraction from the lichen R. farinacea with broad spectrum antiviral activity against DNA viruses (adenovirus and HSV-1) and RNA viruses (HIV-1 and RSV). The effect against DNA and RNA viruses is mediated by different sub-fractions within R. farinacea.

Potential anti-respiratory syncytial virus lead compounds from Aglaia species.

Although the global prevalence of respiratory syncytial virus (RSV) infection, especially among infants and young children is on the increase, there are only limited therapeutic options for treatment of this disease. Therefore, the search for novel antiviral inhibitors of RSV has become more intensive. In a pilot screening of eighteen compounds from various Aglaia species for anti-RSV activity, we identified dammarenolic acid (ignT1), aglaiol (dupT1) and niloticin (cucT1) as potential anti-RSV compounds, with ignT1 being the most potent. Methylation of ignT1 results in a complete loss of anti-RSV activity. Time of addition studies reveal that both ignT1 and dupT1 target the RSV replication at a post-entry stage, although ignT1 was more potent. Dammarenolic acid (ignT1) was also more cytotoxic than aglaiol (dupT1). By carrying out parallel anti-RSV screening with aphidicolin (a highly cytotoxic diterpenoid) and ignT1, we showed that although aphidicolin was more cytotoxic than ignT1, it had virtually no anti-RSV activity. Therefore, dammarenolic acid, aglaiol and niloticin demonstrate potent anti-RSV activity that shouldbe explored further in the current search for anti-RSV therapeutic agents.

Prenylation enhances cytotoxicity of apigenin and liquiritigenin in rat H4IIE hepatoma and C6 glioma cells.

Antioxidative as well as cytotoxic effects of the prenylated flavonoids licoflavone C (8-prenylapigenin) and isobavachin (8-prenylliquiritigenin) were investigated in comparison to the corresponding non-prenylated flavonoids (apigenin, liquiritigenin) and vitexin (apigenin-C8-glucoside) using metabolically active H4IIE hepatoma and metabolically poorly active C6 glioma cells. None of the substances showed radical scavenging activities in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-assay nor were they effective in protection against H2O2-induced intracellular 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation (fluorescent probe for oxidative stress) in H4IIE and C6 cells. When the intrinsic effects of the substances were investigated, licoflavone C and isobavachin exerted a pronounced toxicity in both H4IIE (IC50 values of 42+/-5 and 96+/-19 micromol/L) and C6 cells (IC50 values of 37+/-6 and 69+/-3 micromol/L) while the non-prenylated analogues as well as the glycosylated derivate vitexin showed almost no cytotoxic effect up to 250 micromol/L. In H4IIE cells the induction of apoptotic cell death by licoflavone C and icobavachin was detected as an activation of caspase 3/7 (6- and 3.3-fold, respectively). Based on these experiments we suggest that C8-prenylation of a flavonoid enhances the cytotoxicity inducing an apoptotic cell death in H4IIE cells without affecting antioxidative properties.

Sequestration and possible role of dietary alkaloids in the sponge-feeding mollusk Tylodina perversa.

Opisthobranchs of the genus Tylodina are found at exceedingly distant geographical regions in the marine environment but are always associated with sponges of the order Verongida (e.g., Aplysina species) which serve as prey for these gastropods. We investigated the chemical ecology of the Mediterranean species T. perversa that commonly feeds on A. aerophoba. The gastropod sequesters a set of sponge-derived brominated isoxazoline alkaloids which are accumulated in the mantle and egg masses and are furthermore exuded as part of the mucus when the animal is molested. Based on the documented feeding deterrent properties of the sponge alkaloids against fish, it is speculated that the sequestered sponge alkaloids serve also as a defense for T. perversa. Interestingly, specimens of T. perversa that were either collected while feeding on A. aerophoba or had been kept on these sponges under controlled conditions for several weeks almost always contained the brominated alkaloid aerothionin, which is not detected in A. aerophoba but occurs in the sibling species A. cavernicola instead. The latter sponge is also accepted as a food source by the gastropod, at least under experimental conditions. The possible origin of aerothionin in T. perversa is discussed.

Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells.

Resveratrol (trans-3,5,4',-trihydroxystilbene) is assumed to possess cancer-preventive and cancer-therapeutic properties. The aim of this project was to analyze cellular effects of resveratrol in metabolically active H4IIE rat hepatoma cells in comparison to metabolically poorly active C6 rat glioma cells. Resveratrol is rapidly taken up by both cell types and acts as a potent intracellular antioxidant. On the other hand, resveratrol in higher concentrations is relatively toxic to both cell lines as measured by the neutral red accumulation assay. In H4IIE cells, resveratrol concentrations rapidly decline to very low levels during the first hours of incubation due to formation of resveratrol glucuronides. The first resveratrol effect found at 3h after the start of resveratrol treatment was the induction of mild DNA damage as detected by the comet assay. Cell death was caused via induction of apoptosis as detected by caspase activation, oligonucleosomal DNA fragmentation and formation of apoptotic nuclei. Following DNA damage, resveratrol led to an activation of caspases 2 and 8/10 at 6h and consequently of caspase 3 at 12h, but failed to activate caspase 9. In contrast to H4IIE cells, resveratrol is not metabolised in C6 glioma cells and accumulates to concentrations which are assumed to drive the cell into necrosis. This suggests that the mode of cell death caused by resveratrol and the usefulness of resveratrol for cancer prevention and treatment critically depends on the metabolic capacity of the tumor cell to be eradicated.

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.

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.

Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells.

Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250 microM, 24 h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6h, while caspase-2 and -3/7 are activated after 12 h. After 24 h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3h with 100 microM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage.

Bioactive natural products from marine invertebrates and associated fungi.

Marine natural products with their unique structural features and pronounced biological activities continue to provide lead structures in the search for new drugs from nature. Invertebrates such as sponges, tunicates, mollusks and others that are either sessile or slow moving and mostly lack morphological defense structures have so far provided the largest number of marine-derived secondary constituents including some of the most interesting drug candidates. This review highlights recent research findings of our group related to natural products from marine invertebrates. Areas that are covered include ecological functions of secondary constituents from sponges against predatory fish, the search for new pharmacologically active constituents from sponges and tunicates, and sponge-associated fungi as an evolving source for new bioactive natural products.

Anti-proliferative effects of new staurosporine derivatives isolated from a marine ascidian and its predatory flatworm.

Nine indolocarbazole alkaloids of the staurosporine type, including three new derivatives, were evaluated for their potential as inhibitors of cell proliferation and macromolecule synthesis. Four derivatives were tested as inhibitors of cell proliferation with twelve human leukemia cell lines and demonstrated powerful antiproliferative activities, with 3-hydroxystaurosporine being the most potent. IC(50) values were determined using the cell line MONO-MAC-6 and with an IC(50) of 13 ng/ml, 3-hydroxystaurosporine turned out to be one of the most active staurosporine-type inhibitors described so far. All derivatives, except 3-hydroxy-3'-demethoxy-3'-hydroxystaurosporine and 4'-N-methylstaurosporine very strongly reduced RNA and DNA synthesis with 3-hydroxystaurosporine again being the strongest inhibitor. Analysis of structure-activity relationships demonstrated that hydroxylation of staurosporine at position 3 of the indolocarbazole moiety caused an increase in anti-proliferative activity, while hydroxylation at carbon 11 resulted in a decrease in activity. Our results suggest that not only the presence or absence of hydrophilic substitutions, but also the position of the alteration within the molecule, is important in the antiproliferative properties of the various staurosporine analogues.

The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila.

The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila. In this study, we correlated the pigment production of two lly-positive L. pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant. The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD). This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment. One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases. By screening a genomic library of L. pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin (lly), we identified two recombinant E. coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence. DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene. The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA. Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence.

A cyanogenic glycoside from Canthium schimperianum.

A new cyanogenic glycoside esterified with an iridoid glycoside, 2R-[(2-methoxybenzoylgenoposidyl)-5-O-beta-D-apiofuranosyl-( 1-->6)-beta-glucopyranosyloxy]-2-phenyl acetonitrile, was isolated from the seeds of Canthium schimperianum and identified from its spectroscopic data. The new compound showed weak growth retarding activity towards neonate larvae of Spodoptera littoralis (ED50 8580 ppm) compared to the co-occurring hydroxycinnamic acids, 3,4-dicaffeoylquinic acid (ED50 550 ppm) and 3,5-dicaffeoylquinic acid (ED50 520 ppm).

Insecticidal rocaglamide derivatives from Aglaia spectabilis (Meliaceae).

Bark of Aglaia spectabilis collected on the island of Phu Quoc (Vietnam) yielded insecticidal cyclopentatetrahydrobenzofurans of the rocaglamide type including four new natural products. Structure elucidation of the new compounds is described. All rocaglamide derivatives isolated exhibited strong insecticidal activity towards neonate larvae of the polyphagous pest insect Spodoptera littoralis when incorporated into an artificial diet. LC50 values varied from 0.8 to 80 ppm. The most active compounds isolated, methylrocaglate and C-3' hydroxylmethylrocaglate, were similar with regard to their insecticidal activity to the well-known natural insecticide azadirachtin.

Defensive roles for secondary metabolites from marine sponges and sponge-feeding nudibranchs.

In the marine environment sponges (Porifera) constitute one of the most interesting sources of bioactive natural products. The high frequency of bioactive components in these primitive filter-feeders is interpreted as chemical defence of sponges against environmental stress factors such as predation, overgrowth by fouling organisms or competition for space. The highest incidence of toxic or deterrent sponge metabolites is consequently found in habitats such as coral reefs that are characterized by intense competition and feeding pressure due, for example, to carnivorous fish. Further support for the adaptive significance of sponge constituents is derived from the observation that sponges which are growing exposed are usually more toxic than those growing unexposed. Whereas the chemical defence of sponges seems to be highly effective against most species of fish, a group of shell-less gastropods, the nudibranchs, has specialized on sponges. While feeding on sponges the nudibranchs sequester the effective chemical armoury of their prey, which is subsequently employed for their own protection. Some nudibranchs, however, have become independent of this interspecific flow of natural products and are able to accumulate defensive compounds through de novo synthesis.

A new calyculin derivative from the sponge Theonella swinhoei is a novel and potent inhibitor of tumor cell proliferation.

A novel calyculin derivative was isolated from the marine sponge Theonella swinhoei. Using human and animal tumor cell lines and freshly explanted peripheral blood cells, we investigated several biological effects of this natural product (i.e. cell growth, cytotoxicity, induction of differentiation and apoptosis). The new calyculin exhibited a dose-dependent cytotoxicity against various cell lines from different species and tissues. The ID50 values ranged between 20 and 90 ng/ml. Viability of a multidrug resistant HELA subclone was not affected. Apoptosis of the Hodgkin's lymphoma cell line HDLM-2 induced by antiserum was not prevented by the substance. A reduced drug sensitivity of the monocytic cell line MONOMAC-6 could be observed after induction of differentiation of these cells by phorbol ester and lipopolysaccharide. Even so, non-dividing peripheral blood cells were also resistant to the action of the calyculin derivative, suggesting that the cytotoxin may act preferentially on proliferating cells rather than on quiescent cells. Our data introduce a new calyculin as a marine natural product with interesting features stimulating further studies as a chemotherapeutic or investigational drug.

Do secondary substances in the thallus of a lichen promote CO2 diffusion and prevent depression of net photosynthesis at high water content?

Many lichens show seriously depressed net photosynthesis (NP) at high thallus water contents due to increased carbon dioxide diffusion resistance through blockage of diffusion pathways by water. The soil lichen Diploschistes muscorum, however, shows no depression and NP is close to maximal even at the highest thallus water content. We investigated whether lichen substances (lecanoric and diploschistesic acids) in the cortex and medulla contributed to this ability to maintain high NP. Dry thalli were extracted with water-free acetone and, after this treatment, were found to be fully viable to the extent of continued growth after replanting in the field. No differences were found in the response of NP to thallus water content between the normal and extracted thalli, in fact the response curves were often nearly identical. Thus, in this species it seems that lichen substances did not maintain the water-free diffusion pathways and some other explanation, possibly structural, needs to be sought.

Staurosporine derivatives from the ascidian eudistoma toealensis and its predatory flatworm pseudoceros sp

Two new indolocarbazole alkaloids, 3-hydroxy-3'-demethoxy-3'-hydroxystaurosporine (5) and 11-hydroxy-4'-N-demethylstaurosporine (6), were isolated from the marine ascidian Eudistoma toealensis and its predator, the marine flatworm Pseudoceros sp. In addition, five known derivatives were isolated in their protonated states, which caused the pyran-ring system to adopt a boat conformation. The structures were determined by 1D and 2D homonuclear and (1)H-detected heteronuclear NMR spectroscopy and from comparisons with published data. The heteronuclear correlations were necessary to establish reliable data for the structure elucidation.