Ren Xiang Tan

Biology and chemistry of endophytes

This review focuses on new endophyte-related findings in biology and ecology, and also summarises the various metabolites isolated from endophytes.

New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua

In addition to ergosterol (I), 3 beta,5 alpha,6 beta-trihydroxyergosta-7,22-diene (II), 3 beta-hydroxy-ergosta-5-ene (III), 3-oxo-ergosta-4,6,8(14),22-tetraene (TV), 3 beta-hydroxy-5 alpha,8 alpha-epidioxy-ergosta-6,22-diene, 3 beta-hydroxy-5 alpha,8 alpha-epidioxy-ergosta-6,9(11),22-triene (VI) and 3-oxo-ergosta-4-ene (VII), a plant hormone indole-3-acetic acid (IAA) and three new antimicrobial metabolites were characterized from the culture of Colletotrichum sp., an endophyte isolated from inside the stem of Artemisia annua. The structures of the new metabolites were elucidated by a combination of spectroscopic methods (IR, MS, H-1 and C-13 NMR) as 6-isoprenylindole-3-carboxylic acid (1), 3 beta,5 alpha-dihydroxy-6 beta-acetoxy-ergosta-7,22 (2) and 3 beta,5 alpha-dihydroxy-6 beta-phenylacetyloxy-ergosta-7,22-diene (3), respectively. The compounds 1-3 and III-V inhibited the growth of all the tested bacteria (Bacillus subtilis, Staphylococcus aureus, Sarcina lutea and Pseudomonas sp.) with minimal inhibitory concentrations (MICs) ranging from 25 to 75 mu g/ml. Moreover, metabolites 2 and 3, together with the known sterols III and V, were inhibitory against the fungi Candida albicans and Aspergillus niger with MICs between 50 and 100 mu g/ml. At 200 mu g/ml, compounds 1-3, III and IV were shown to be fungistatic to the crop pathogenic fungi Gaeumannomyces graminis var, tritici, Rhizoctonia cerealis, Helminthosporium sativum and Phytophthora capisici. This is the first report on the endophytic fungus from A. annua and the bioactive metabolites thereof

Inhibition of xanthine oxidase by some Chinese medicinal plants used to treat gout.

The enzyme xanthine oxidase catalyses the oxidation of hypoxanthine to xanthine and then to uric acid, which plays a crucial role in gout. A total of 122 traditional Chinese medicinal plants, selected according to the clinical efficacy and prescription frequency for the treatment of gout and other hyperuricemia-related disorders, have been evaluated for the enzyme inhibitory activity. Among the 122 methanol extracts derived from these species, 69 were shown to be inhibitory at 100 microg/ml, with 29 having greater than 50% inhibition. As to the equal amount of water extracts, 40 were disclosed to be active at 100 microg/ml, with 13 possessing more than 50% inhibition. At 50 microg/ml, 58 methanol and 24 water extracts exhibited inhibitory activity, with 15 of the former and two of the latter showing greater than 50% inhibition. The most active was the methanol extract of the twig of Cinnamomum cassia (Lauraceae) (IC(50), 18 microg/ml), which was followed immediately by those of the flower of Chrysanthemum indicum (Asteraceae) (IC(50), 22 microg/ml) and the leaves of Lycopus europaeus (Lamiatae) (IC(50), 26 microg/ml). Among the water extracts, the strongest inhibition of the enzyme was observed with that of the rhizome of Polygonum cuspidatum (Polygonaceae) (IC(50), 38 microg/ml). The IC(50) value of allopurinol used as a positive control was 1.06 microg/ml. The study demonstrated that the effects for these medicinal plants used for the gout treatment were based, at least in part, on the xanthine oxidase inhibitory action.

Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi.

Artemisia annua, well recognized for its production of antimalarial drug artemisinin, is seldom attacked by any of phytopathogenic fungi, which could be partially associated with the presence of endophytes. Present investigation is aiming at disclosing whether the endophytes inside A. annua produce antifungal substances. A total of 39 endophytes were isolated and fermented, and the ferment broth was evaluated in vitro for the antifungal activity against crop-threatening fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sativum, Fusarium graminearum, Gerlachia nivalis and Phytophthora capsici. These plant pathogens are still causing wheat take-all, sharp eyespot, common rot, scab, snow mould, and pepper phytophthora blight, respectively. Out of 39 endophytes investigated, 21 can produce in vitro substances that are inhibitory to all or a few of the tested phytopathogens whereas the rest yielded nothing active. Moreover, the most active broth of endophyte IV403 was extracted with EtOAc and n-butanol, and comparisons of the antifungal activity of the extracts indicated that the major active metabolites were EtOAc-extractable.

Inhibition of xanthine oxidase by liquiritigenin and isoliquiritigenin isolated from Sinofranchetia chinensis.

Abstract. The methanol extract of the stem of Sinofranchetia chinensis inhibited the activity of xanthine oxidase in vitro. Bioassay-guided purification led to the isolation of liquiritigenin and isoliquiritigenin as the main xanthine oxidase inhibitors. This inhibition of enzyme activity was found to be dose dependent, with an IC50 value of approximately 49.3 μM for liquiritigenin and 55.8 μM for isoliquiritigenin. Lineweaver-Burk transformation of the inhibition data indicated that the inhibition was of a mixed type for both liquiritigenin and isoliquiritigenin. For liquiritigenin, the Ki and KI were determined to be 14.0 μM and 151.6 μM, respectively. For isoliquiritigenin, the Ki and KI were determined to be 17.4 μM and 81.9 μM, respectively. These results suggest that these natural products could be used to treat conditions where the inhibition of xanthine oxidase is warranted.

NF-κB modulators in osteolytic bone diseases

Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-kappaB is a set of nuclear factors that bind to consensus DNA sequences called kappaB sites, and is essential for osteoclast formation and survival. NF-kappaB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-alpha and IL-1, which differentially regulate classical and/or alternative NF-kappaB pathways in osteoclastic cells. These pathways are also modulated by NF-kappaB mediators, including TRAF6, aPKC, p62/SQSTM1 and deubiquitinating enzyme CYLD that are involved in the ubiquitin-proteasome system during RANK-mediated osteoclastogenesis. Abnormal activation of NF-kappaB signalling in osteoclasts has been associated with excessive osteoclastic activity, and frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, Pagets disease of bone, and periodontitis. NF-kappaB modulators such as parthenolide and NEMO-binding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models. Unravelling the structure and function of NF-kappaB pathways in osteoclasts and other cell types will be important in developing new strategies for treatments of bone diseases.

Unprecedented immunosuppressive polyketides from Daldinia eschscholzii, a mantis-associated fungus.

Immunosuppressants are required for an array of medical purposes, such as organ transplantations and the treatment of autoimmune-associated diseases. However, most of the currently available immunosuppressive drugs have been shown to inevitably possess severe adverse effects, such as hepatotoxicity, nephrotoxicity, and hypertension induction. Therefore, there is an urgent need for new therapeutic agents for modulating the autoimmune response. Some microorganisms are a reliable source of immunocompromising compounds, as exemplified by the discovery of cyclosporin A (CsA), rapamycin, and FK506. Symbionts, a diverse microbial community present in plants, insects, and mammals without the generation of any detectable symptoms, are receiving renewed attention for their production of chemically inspiring and biologically potent metabolites, presumably as a result of their long coevolution with hosts. In particular, some insect-associated fungi might have acquired instinctlike capabilities for synthesizing immunoalleviating metabolites from their initial microbe–host interaction through to the final colonization. In continuation of our characterization of new bioactive metabolites from endophyte cultures, this observation tempted us to explore novel immunosuppressive metabolites that could be produced by fungi inhabiting healthy insect organs, such as the mantis gut, which is clearly an important entrance and shelter for symbionts (including quiescent pathogens) and meal-carried “foreign” microbes. As expected, a preliminary screen recognized the presence of an immunosuppressive substance or immunosuppressive substances in a culture of Daldinia eschscholzii IFB-TL01 residing in the gut of the mantis species Tenodera aridifolia, a common predator of many insects that feed on plants harboring endophyte. Subsequent bioassay-guided fractionation of the extract derived from the scaled-up fermentation of the fungus afforded two polyketides, dalesconols A (1) and B

Antidepressant effects of Banxia Houpu decoction, a traditional Chinese medicinal empirical formula.

Banxia Houpu Decoction, having been used for the treatment of depression-related diseases since ancient times, is a traditional Chinese medicinal empirical formula consisting of Pinellia ternata, Poria cocos, Magnolia officinalis, Perilla frutescens and Zingiber officinale. The effects of the total decoction extract and five fractions therefrom, were evaluated in mice by tail suspension and forced swimming tests. The total 90% ethanol extract of the decoction was shown to possess an antidepressant activity that was close to that of Prozac, an antidepressant agent being applied clinically. Furthermore, the active principles were desmonstrated to be mainly in the aqueous (Bx4) and lipophic (Bx5) parts of the decoction extract while the polyphenol fraction (Bx2) exhibited a moderate action.

High-performance liquid chromatographic analysis of bioactive triterpenes in Perilla frutescens

Perilla frutescens (L.) Britt. (Lamiaceae), a famous traditional Chinese medicine, has been used for the treatment of various diseases. To evaluate the quality of P. frutescens, a simple, rapid and accurate high-performance liquid chromatography (HPLC) method was developed for the assessment of three bioactive triterpene acids: tormentic acid (TA), oleanolic acid (OA) and ursolic acid (UA). The HPLC system used an Spherisob octadecylsilyl silica (ODS) column with acetonitrile and aqueous H(3)PO(4) as the mobile phase and detection at 206 nm. The method was precise with relative standard deviations for these three constituents that ranged between 0.6-1.5% (intraday) and 0.7-2.6% (interday). The content of these three phytochemicals in the leaves of P. frutescens growing at eight different locations of China was determined to establish the effectiveness of the method.

Chaetoglobosin U, a Cytochalasan Alkaloid from Endophytic Chaetomium globosum IFB-E019

A new cytotoxic cytochalasan-based alkaloid named chaetoglobosin U (1), along with four known analogues, chaetoglobosins C (2), F (3), and E (5) and penochalasin A (4), has been characterized from the EtOAc extract of a solid culture of Chaetomium globosum IFB-E019, an endophytic fungus residing inside the stem of healthy Imperata cylindrica. The structure of chaetoglobosin U was determined through correlative analyses of its UV, IR, CD, MS, and 1D ((1)H and(13)C NMR and DEPT) and 2D NMR (COSY, NOESY, HMQC, and HMBC) data. Chaetoglobosin U (1) exhibited cytotoxic activity against the human nasopharyngeal epidermoid tumor KB cell line with an IC(50) value of 16.0 microM, comparable to that (14.0 microM) of 5-fluorouracil co-assayed as a positive reference. The known analogues 2-5 were moderately active to the cell line, with IC(50) values of 34.0, 52.0, 48.0, and 40.0 microM, respectively.