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...

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Curcuma longa L. (Zingiberaceae) is a well-known turmeric rhizome whose main chemical constituent curcumin (1) has a potent antioxidant effect.1) However, it also possesses a distinct yellow color, which limits its use.We have been studying a microbial conversion of natural products [2][3][4] by hypothesizing that the enzyme system in cells of endophytic microbes 5,6) can be activated to convert the chemical constituents of the host plant since endophytic microbes live near the chemical constituents of the plant. In this paper, we report on the conversion of curcumin (1) by Diaporthe sp., an endophytic fungus isolated from the rhizome of Curcuma longa, into four colorless hydroderivatives including a novel substance, named neohexahydrocurcumin (3).Thirteen endophytic filamentous fungi (CLO-1-CLO-13) were obtained from the rhizome of Curcuma longa (Zingiberaceae) using a previously reported procedure 7,8) and subjected to the base sequence analysis of the internal transcribed spacer-1 (ITS-1), 5.8S ribosomal DNA (rDNA), and ITS-2 region. The analysis showed that the fungi comprised three Diaporthe spp. (CLO-3, CLO-4, and CLO-13), five Phomopsis spp. (CLO-6, CLO-9, CLO-10, CLO-11, and CLO-12), two Neofuicoccum spp. (CLO-1 and CLO-8), one Botryosphaeria sp. (CLO-7), one Gibberella sp. (CLO-2), and one Sarcinomyces sp. (CLO-5). Then, endophytic microbes for the microbial conversion of 1 were selected through several preliminary experiments, which led to the selection of the endophytic fungus Diaporthe sp. CLO-13 (registered as AB537342 in DDBJ/EMBL/GeneBank).CLO-13 was inoculated into 2% glucose-yeast extractpeptone medium. After cultivation at 28°C with rotary shaking at 90 rpm for 7 d, a methanol solution of curcumin (1) was added to the culture, which was further cultivated for 2 d. The cultivation medium including the fungal bodies was homogenized and extracted with chloroform. The chloroform-soluble portion was separated by silica gel column chromatography to give a colorless curcuminoid fraction, which was purified by reversed-phase HPLC to afford the following products in their respective yields: 2, 18%; 3, 27%; 4, 8%; and 5, 17%.Compounds 4 and 5 were white powders and showed a quasi-molecular ion peak at m/z 375 corresponding to C 21 H 27 O 6 by negative FAB-MS. From the physicochemical properties obtained by IR, UV, 1 H-NMR, 13C-NMR measurements, both 4 and 5 were identified as be 1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane-3,5-diol (octahydrocurcumin). 9)Furthermore, the optical rotation data showed that absolute structure of 4 was (3S,5S)-octahydrocurcumin ([a] D Ϫ8. 3°, lit. 9) [a] D Ϫ7.4°) and 5 was meso-octahydrocurcuminCompound 2, a white powder, showed a quasi-molecular ion peak at m/z 371 corresponding to C 21 H 23 O 6 by negative FAB-MS. Its IR spectrum indicated the presence of hydroxyl and aromatic functional groups and its UV spectrum showed Gakuen-cho, Fukuyama, Hiroshima 729-0292, Japan: and c Rohokeimeido Co., Ltd.; 579-25 Kusugawa, Kamiyaku-cho, Kumage-gun, Kagoshima 891-4206, Japan. ...

We report that the endophytic filamentous fungus Diaporthe sp., isolated from Cinchona ledgeriana and cultivated in a synthetic liquid medium, produces Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine). This shows that Cinchona alkaloids are produced not only in Cinchona plant cells, but also in endophytic microbe cells.

We report that an endophytic filamentous fungus species of the genus Diaporthe isolated from Cinchona ledgeriana (Rubiaceae) produces Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine) upon cultivation in a synthetic liquid medium. This study provides evidence that Cinchona alkaloids are produced not only in Cinchona plant cells, but also in the endophytic microbe cells, and will help to elucidate the relationship between endophytic microbes and their host plants.Key words Cinchona alkaloid; endophyte; Cinchona ledgeriana; Diaporthe sp.; bioproduction; quinine Endophytes, which are defined as microbes that live either symbiotically or parasitically inside the plant body, have attracted much research interest.1-4) However, their biological roles have not yet been entirely clarified. We are interested in the relationship between the chemical constituents of a plant and the endophytic microbes within the same plant. So far, we have reported the bioconversion of Cinchona alkaloids into their 1-N-oxide derivatives by Xylaria species associated with Cinchona pubescens, 5) the stereoselective oxidation of catechins into 3,4-cis-dihydroflavan derivatives by Diaporthe species associated with the tea plant Camellia sinensis, 6,7) and the bioconversion of curcumin into colorless hydroderivatives by Diaporthe species associated with Curcuma longa. 8)Cinchona alkaloids such as quinine, quinidine, cinchonidine, and cinchonine 9-11) have been regarded as the chemical constituents of Cinchona plants since their isolation from Cinchona bark in 1820 by Pelletier and Caventou.12,13) Among these alkaloids, quinine is a well-known antimalarial drug effective against the erythrocyte stage of the parasite Plasmodium falciparum.14) Here, we report the bioproduction of Cinchona alkaloids by endophytic microbes associated with the Cinchona plant. We chose Cinchona ledgeriana MOENS ex TRIMEN 15) (Rubiaceae) as the host plant for study because it is thought to contain the highest amounts of Cinchona alkaloids within the genus Cinchona.In total, we isolated 21 endophytic filamentous fungi from the young stems of Cinchona ledgeriana collected in the Puncak district, West Java, Indonesia. These fungi were classified as belonging to nine Phomopsis spp., six Diaporthe spp., two Schizophyllum spp., two Penicillium spp., one Fomitopsis sp. and one Arthrinium sp. by nucleotide sequence analysis of the internal transcribed spacers (ITS-1 and ITS-2) including 5.8S ribosomal DNA region. 16) In the present paper, 17) we focus on one filamentous fungus species of Diaporthe (CLF-J: registered as AB505415 in DDBJ/EMBL/GeneBank). A synthetic liquid medium (3.0 g of D-glucose, 1.0 g of D-mannose, 0.2 g of Bacto tryptic soy broth, and 1000 mL of tap water) was chosen as cultivation medium after several preliminary experiments.The fungus was transferred from potato dextrose agar (PDA) slant medium onto water agar medium and grown for about 10 d to standardize cultivation and to remove unnecessary factors. The fungus was inoculated into the...

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Four kinds of endophytic filamentous fungi (code names: CLS-1, CLS-2, CLS-3, and CLS-4) associated with the seeds of Cinchona ledgeriana (Rubiaceae) from West Java, Indonesia, were isolated. All of the isolates were classified into Diaporthe spp. based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) including the 5.8S ribosomal DNA region. All four of these endophytic fungi produce Cinchona alkaloids, mainly quinine and quinidine, in synthetic liquid medium.

We have investigated the ability of endophytic filamentous fungi associated with Cinchona ledgeriana (Rubiaceae) to produce Cinchona alkaloids on potato dextrose agar medium and in a synthetic liquid medium. It was found that all twenty-one endophytic fungi produce Cinchona alkaloids, despite their genetic differences.