Transformation of Cinchona Alkaloids into 1-N-Oxide Derivatives by Endophytic Xylaria sp. Isolated from Cinchona pubescens.

Abstract

Cinchona alkaloids have been used as drugs for the treatment of several diseases. Quinine is much popular as an antimalarial drug against the erythrocyte stage of the parasite. However, to the best of our knowledge, no research on the endophytes isolated from Cinchona plants has been reported. We predicted that endophytic microbes living in Cinchona plants would transform Cinchona alkaloids into their chemical derivatives. In the present paper, we report the microbial transformation of Cinchona alkaloids by the endophytic fungus Xylaria sp. which was isolated from the young stems of Cinchona pubescens VAHL. (Rubiaceae). 1)The young stems of Cinchona pubescens collected in West Java, Indonesia, were cut into pieces ca. 1 cm in length and the exterior was sterilized with 70% EtOH and 5.3% sodium hypochlorite. Then the pieces were placed on corn-meal malt agar (CMMA) containing chroramphenicol and incubated at 27°C for 3 d. Then individual colonies on the plates were transferred to potato dextrose agar (PDA) and incubated again at 27°C for 5 d with periodic checks of purity to obtain a total of 10 endophytic filamentous fungi.Through several screening tests for the microbial transformation of Cinchona alkaloids by the 10 endophytic fungi, it was found that the filamentous fungus Xylaria sp.2) transforms four Cinchona alkaloid hydrochloride salts, i.e., quinine hydrochloride (1 · HCl), quinidine hydrochloride (2 · HCl), cinchonidine hydrochloride (3 · HCl), and cinchonine hydrochloride (4 · HCl), into chemical derivatives in potato dextrose broth (PDB) medium.Each cultivation medium including the fungus bodies was homogenized and extracted with CHCl 3 . The CHCl 3 -soluble portion was separated by HPLC on Hibar LiChrosorb NH 2 to afford 1a (90% yield) from 1 · HCl, 2a (71% yield) from 2 · HCl, 3a (82% yield) from 3 · HCl, and 4a (52% yield) from 4 · HCl, respectively. Among those transformation products, 1a, 2a, and 3a were identified by comparisons of their physicochemical data (mp, [a] D , 1 H-, 13 C-NMR, IR, and UV) with those of quinine 1-N-oxide, 3-5) quinidine 1-N-oxide, [3][4][5][6] and cinchonidine 1-N-oxide, 3,7) respectively. 4a, which was transformed from cinchonine hydrochloride (4 · HCl), showed similar absorptions to those of 4 in the IR and UV spectra. The 13 C-NMR spectrum (in CD 3 OD) also showed similar signal patterns to those of 4, except the chemical shifts of 2-C (d 65.0), 6-C (d 66.6), and 8-C (d 74.0) observed in lower magnetic fields than those of 2-C (d 50.3), 6-C (d 50.9), and 8-C (d 61.3) for cinchonine (4). From the above findings, it was assumed that 4a might be cinchonine 1-N-oxide, which was previously prepared from 4 by Dodin et al. 7)The endophytic fungus Xylaria sp. was submitted to DNA analysis of the 18S, ITS-1, 5.8S, and ITS-2 rDNA regions. Comparison of the base sequences with those of the authentic Xylaria enteroleuca (CBS 651.89),8) analyzed by us, showed 99.9% similarity for the 18S region [X. enteroleuca (CBS 651.89): 3 gaps/2455 bp] and 100% similarity for the ITS1-5.8S-IT...

Bisanthraquinone Metabolites Produced by the Endophytic Fungus Diaporthe sp.

“…The IR spectrum exhibited the absorptions due to hydroxyl, carbonyl and aromatic functions and the UV spectrum showed the characteristic absorption pattern of anthraquinone derivatives. The complete decoupled 13 C-NMR of 1 showed totally fifteen carbon signals, which is only half the number of the molecular formula determined by the high-resolution FAB-MS. Therefore, it has been found that 1 is a bis-type compound.…”

Plant Bioactive Metabolites and Drugs Produced by Endophytic Fungi of Spermatophyta

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Current Understanding of Endophytes: Their Relevance, Importance, and Industrial Potentials

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“…Endophytic bacteria have improved wheat growth through production of phytohormones ; increased cotton disease resistance; increased rice production by increasing mineral availability; fix nitrogen in wheat and rice; decrease propensity to frost damage; contribute to increase potato tuber formation and corn pest management under heat stress conditions. [113]. An artistic rendition according to [52].…”