Endophyte composition and Cinchona alkaloid production abilities of Cinchona ledgeriana cultivated in Japan.

New eight endophytic filamentous fungi were isolated from the young stems of Cinchona ledgeriana (Rubiaceae) cultivated in Japan. They were classified into four genera based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2), including the 5.8S ribosomal DNA region. Of the eight fungi isolated, there were five genera Cladosporium, one Meira sp., one Diaporthe sp. and one Penicillium sp. Genus of Cladosporium and Meira were first isolated fungi from Cinchona plant. In a previous study, we applied the same process to the same plant cultivated in Indonesia. The endophyte compositions for the two cultivation regions were found to differ at the genera level. The ability of Cinchona endophytes cultivated in Japan to produce Cinchona alkaloids was also assessed. We found that three isolates have producing ability of Cinchona alkaloids. However, the amount produced was very small compared to that produced by the endophytes of Indonesian Cinchona ledgeriana. In addition, the total content amount of Cinchona alkaloids, especially quinine, produced by the extract of Cinchona cultivated in Japan was much smaller than that from Indonesia. These finding indicate that endophyte composition has an influence on the Cinchona alkaloid content amount in the Cinchona ledgeriana host.

Cinchona alkaloids from Cinchona succirubra and Cinchona ledgeriana.

Seven new cinchona alkaloids, cinchonanines A-G (1-7), and 29 known alkaloids were isolated from the barks of Cinchona surrirubra and C. ledgeriana collected from Yunnan Province in China. The new structures were elucidated by extensive spectroscopic analysis. All compounds were evaluated for their cytotoxicity against five human cancer cell lines. Compounds 2, 13, 14, and 15 showed moderate cytotoxicity.

Extraction of alkaloids for NMR-based profiling: exploratory analysis of an archaic Cinchona bark collection.

A museum collection of Cinchonae cortex samples (n = 117), from the period 1850-1950, was extracted with a mixture of chloroform-d1, methanol-d4, water-d2, and perchloric acid in the ratios 5 : 5 : 1 : 1. The extracts were directly analyzed using 1H NMR spectroscopy (600 MHz) and the spectra evaluated using principal component analysis (PCA) and total statistical correlation spectroscopy (STOCSY). A new method called STOCSY-CA, where CA stands for component analysis, is described, and an analysis using this method is presented. It was found that the samples had a rather homogenous content of the well-known cinchona alkaloids quinine, cinchonine, and cinchonidine without any apparent clustering. Signals from analogues were detected but not in substantial amounts. The main variation was related to the absolute amounts of extracted alkaloids, which was attributed to the evolution of the Cinchona tree cultivation during the period in which the samples were collected.

Potent protecting effects of Catuaba (Anemopaegma mirandum) extracts against hydroperoxide-induced cytotoxicity.

Ishigami et al. (Ishigami et al., 1998) reported that squalene monohydroperoxide (SQOOH) induced skin damage in hairless mice. Kohno and Takahashi (Kohno and Takahashi, 1993) reported that SQOOH induced cytotoxicity against Chinese hamster lung fibroblasts. We have already evaluated the efficacy of extracts obtained from Brazilian herbal medicines in protecting the normal human epidermis keratinocytes [NHEK(B)] against the cytotoxicity caused by SQOOH. The EtOAc extract was separated by silica-gel column chromatography into eight fractions. Fractions (Fr) 1,3 and 5 significantly protected rat basophilic leukemia (RBL-2H3) cells from the release of beta-hexosaminidase due to SQOOH. Additionally, Fr5-1 was most effective in a Gunze three-dimensional cultured human skin model (Vitrolife-skin) against the cytotoxicity due to SQOOH and the release of interleukin (IL)-2 and IL-4. The mixture of cinchonains Ia and Ib and the mixture of cinchonains IIa and IIb were isolated from Fr3 and Fr5-1, respectively. The results suggest that the addition of SQOOH caused the reduction in cell viability and the release of beta-hexosaminidase and cytokines as chemical mediators. The extract of Catuaba (Anemopaegma mirandum) prevented these toxic effects with the main active agents suggested to be cinchonains IIa and IIb.

HPTLC method for the estimation of alkaloids of Cinchona officinalis stem bark and its marketed formulations.

We report a sensitive method for the estimation of quinine (Qn), cinchonine (Cn), and cinchonidine (Cnd) and a new method based on fluorescence enhancement and detection and quantification of quinidine (Qnd) from Cinchona stem bark and its formulations, using HPTLC. Standard solutions of Qn, Qnd, Cn, and Cnd were applied on precoated HPTLC plates and developed with chloroform/diethylamine (9.6:1.4 v/v). The plates were scanned and quantified at 226 nm for Qn, Cn, Cnd and for Qnd at 366 nm in fluorescence and reflectance mode ([symbol: see text] K400 filter). The method was validated for precision, accuracy and repeatability. Further, the stem bark of Cinchona officinalis and some herbal and homeopathic formulations were evaluated for their individual alkaloid content applying the developed method.

Automated multiple development thin layer chromatography of some plant extracts.

The separation of ten plant extracts using automated multiple development thin-layer chromatography (AMD -TLC) is described. Alcoholic extracts were obtained from Cinchona succirubra, Aesculus hippocastanum, Berberis vulgaris. Artemisia abrotanum, Carduus marianus, Thuja occidentalis, Baptisia tinctoria, Paulinia cupana, Lycopus europaeus and Echinacea angustifolia. The separation was performed on silica plates (Sil G-50 UV 254 (Macherey-Nagel), 10 x 20 cm). AMD was achieved in 25 steps using methanol, ethyl acetate, toluene, 1,2-dichloroethane, 25% ammonia solution and anhydrous formic acid as modifiers. The chromatograms were evaluated with a Shimadzu CS-9000 dual-wavelength flying-spot scanner. Better separations were obtained using AMD than isocratic elution.