Novel nikkomycin analogues generated by mutasynthesis in Streptomyces ansochromogenes.

BACKGROUND: Nikkomycins are competitive inhibitors of chitin synthase and inhibit the growth of filamentous fungi, insects, acarids and yeasts. The gene cluster responsible for biosynthesis of nikkomycins has been cloned and the biosynthetic pathway was elucidated at the genetic, enzymatic and regulatory levels. RESULTS: Streptomyces ansochromogenes ΔsanL was constructed by homologous recombination and the mutant strain was fed with benzoic acid, 4-hydroxybenzoic acid, nicotinic acid and isonicotinic acid. Two novel nikkomycin analogues were produced when cultures were supplemented with nicotinic acid. These two compounds were identified as nikkomycin Px and Pz by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Bioassays against Candida albicans and Alternaria longipes showed that nikkomycin Px and Pz exhibited comparatively strong inhibitory activity as nikkomycin X and Z produced by Streptomyces ansochromogenes 7100 (wild-type strain). Moreover, nikkomycin Px and Pz were found to be more stable than nikkomycin X and Z at different pH and temperature conditions. CONCLUSIONS: Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by mutasynthesis with the sanL inactivated mutant of Streptomyces ansochromogenes 7100. Although antifungal activities of these two compounds are similar to those of nikkomycin X and Z, their stabilities are much better than nikkomycin X and Z under different pHs and temperatures.

Rapid isolation and purification of inotodiol and trametenolic acid from Inonotus obliquus by high-speed counter-current chromatography with evaporative light scatting detection.

INTRODUCTION: In Eastern Europe, especially Russia, the fruiting body of Inonotus obliquus has been used as a folk medicine for cancer since the sixteenth or seventeenth century. Inotodiol and trametenolic acid are considered to be the main bioactive compounds of the fruiting body of the mushroom. These compounds show various biological activities, including anti-tumour, anti-viral, hypoglycaemic, anti-oxidant and cyto-protective. However, effective methods for isolating and purifying inotodiol and trametenolic acid from the fruiting body of Inonotus obliquus are not currently available. OBJECTIVE: To develop a suitable preparative method in order to isolate inotodiol and trametenolic acid from a complex Inonotus obliquus extract by preparative high-speed counter-current chromatography (HSCCC). METHODOLOGY: Inotodiol and trametenolic acid were rapidly isolated and purified from the chloroform extract of Inonotus obliquus (Fr.) by HSCCC with evaporative light scatting detection (ELSD). The purity of the obtained target compounds was analysed by high-performance liquid chromatography (HPLC) with ELSD. The structures of the two compounds were identified by ¹H NMR and ¹³C NMR. RESULT: The target compounds were finally isolated and purified with a solvent system of hexane:ethyl acetate:methanol:water (1:0.4:1:0.4, v/v/v/v). In a single operation, 100 mg of the I. obliquus extracts yielded 13.0 mg of inotodiol and 7.0 mg of trametenolic acid. The entire separation and purification process took less than 5 h. The purities of obtained inotodiol and trametenolic acid were 97.51 and 94.04%, respectively. CONCLUSION: HSCCC-ELSD was an efficient and rapid method for the separation and purification of inotodiol and trametenolic acid from I. obliquus.