First observation on the predation of a non-arthropod species by a dung beetle species: The case of Canthon chalybaeus and the snail Bulimulus apodemetes.

We described, for the first time, a case of predation of a non-arthropod species by a dung beetle species. Canthon chalybaeus Blanchard, 1843 kills healthy individuals of the terrestrial snail Bulimulus apodemetes (D'Orbigny, 1835) showing an evident pattern of physical aggressiveness in the attacks using the dentate clypeus and the anterior tibiae. The description of this predatory behaviour was complemented with the analysis of the chemical secretions of the pygidial glands of C. chalybaeus, highlighting those main chemical compounds that, due to their potential toxicity, could contribute to death of the snail. We observed a high frequency of predatory interactions reinforcing the idea that predation in dung beetles is not accidental and although it is opportunistic it involves a series of behavioural sophistications that suggest an evolutionary pattern within Deltochilini that should not only be better studied from a behavioural point of view but also phylogenetically.

Isolation and Antibacterial Activity of Indole Alkaloids from Pseudomonas aeruginosa UWI-1.

In this study, we report the first isolation of three antibiotic indole alkaloid compounds from a Pseudomonad bacterium, Pseudomonas aeruginosa UWI-1. The bacterium was batch fermented in a modified Luria Broth medium and compounds were solvent extracted and isolated by bioassay-guided fractionation. The three compounds were identified as (1) tris(1H-indol-3-yl) methylium, (2) bis(indol-3-yl) phenylmethane, and (3) indolo (2, 1b) quinazoline-6, 12 dione. A combination of 1D and 2D NMR, high-resolution mass spectrometry data and comparison from related data from the literature was used to determine the chemical structures of the compounds. Compounds 1-3 were evaluated in vitro for their antimicrobial activities against a wide range of microorganisms using the broth microdilution technique. Compounds 1 and 2 displayed antibacterial activity against only Gram-positive pathogens, although 1 had significantly lower minimum inhibitory concentration (MIC) values than 2. Compound 3 displayed potent broad-spectrum antimicrobial activity against a range of Gram positive and negative bacteria. Several genes identified from the genome of P. aeruginosa UWI-1 were postulated to contribute to the biosynthesis of these compounds and we attempted to outline a possible route for bacterial synthesis. This study demonstrated the extended metabolic capability of Pseudomonas aeruginosa in synthesizing new chemotypes of bioactive compounds.

Bioinspired biomolecules: Mycosporine-like amino acids and scytonemin from Lyngbya sp. with UV-protection potentialities.

Since the beginning of life on Earth, cyanobacteria have been exposed to natural ultraviolet-A radiation (UV-A, 315-400 nm) and ultraviolet-B radiation (UV-B, 280-315 nm), affecting their cells' biomolecules. These photoautotrophic organisms have needed to evolve to survive and thus, have developed different mechanisms against ultraviolet radiation. These mechanisms include UVR avoidance, DNA repair, and cell protection by producing photoprotective compounds like Scytonemin, carotenoids, and Mycosporine-like amino acids (MAAs). Lyngbya marine species are commercially important due to their secondary metabolites that show a range of biological activities including antibacterial, insecticidal, anticancer, antifungal, and enzyme inhibitor. The main topic in this review covers the Lyngbya sp., a cyanobacteria genus that presents photoprotection provided by the UV-absorbing/screening compounds such as MAAs and Scytonemin. These compounds have considerable potentialities to be used in the cosmeceutical, pharmaceutical, biotechnological and biomedical sectors and other related manufacturing industries with an additional value of environment friendly in nature. Scytonemin has UV protectant, anti-inflammatory, anti-proliferative, and antioxidant activity. MAAs act as sunscreens, provide additional protection as antioxidants, can be used as UV protectors, activators of cell proliferation, skin-care products, and even as photo-stabilizing additives in paints, plastics, and varnishes. The five MAAs identified so far in Lyngbya sp. are Asterina-330, M-312, Palythine, Porphyra-334, and Shinorine are capable of dissipating absorbed radiation as harmless heat without producing reactive oxygen species.

Meroditerpene pyrone, tryptoquivaline and brasiliamide derivatives from the fungus Neosartorya pseudofischeri.

Two new meroditerpene pyrones, chevalone F (1) and 11-hydroxychevalone E (2), a new tryptoquivaline analog, tryptoquivaline V (3) and a new brasiliamide analog, brasiliamide G (4), together with thirteen known compounds, chevalones A-C (5-7), chevalone E (8), 11-hydroxychevalone C (9), pyripyropene A (10), isochaetominine C (11), pyrrolobenzoxazine terpenoids CJ-12662 (12) and CJ-12663 (13), fischerindoline (14), eurochevalierine (15), 1,4-diacetyl-2,5-dibenzylpiperazine-3,7''-oxide (16) and lecanorin (17) were isolated from the fungus Neosartorya pseudofischeri. Their structures were established on the basis of spectroscopic evidence. Compound 2 showed weak antibacterial activity against Escherichia coli and Salmonella enterica serovar Typhimurium, whereas compounds 7, 12, 13 and 15 showed antibacterial activity against Bacillus cereus and Staphylococcus aureus. In addition, compounds 13 and 14 showed cytotoxicity against KB and MCF-7 cancer cell lines, as well as the Vero cell line.

Antichagasic effect of violacein from Chromobacterium violaceum.

AIMS: Violacein (VIO), a bacterial pigment produced by Chromobacterium violaceum, was examined to evaluate the antichagasic activity and its action mechanism against Trypanosoma cruzi Y strain. METHODS AND RESULTS: Violacein was tested against the epimastigote, trypomastigote and amastigote forms of T. cruzi Y strain (benznidazole-resistant strain). VIO inhibited all T. cruzi developmental forms, including amastigotes, which is implicated in the burden of infection in the chronic phase of Chagas disease (CD). VIO induced cell death in T. cruzi through apoptosis, as determined by flow cytometry analyses with specific molecular probes and morphological alterations, such as involvement of reactive oxygen species and changes in mitochondrial membrane potential and cell shrinkage. CONCLUSION: The results suggest antichagasic activity of VIO against T. cruzi Y strain with apoptotic involvement. SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of CD has limited efficacy and side effects that restrict patient tolerability and compliance. The VIO molecule could be used as a model for therapeutic alternatives for this disease.

Impact of violacein from Chromobacterium violaceum on the mammalian gut microbiome.

Violacein is a violet pigment produced by Chromobacterium violaceum that possesses several functions such as antibacterial, antiviral, antifungal, and antioxidant activities. The search for potential compounds and therapies that may interfere with and modulate the gut microbial consortia without causing severe damage and increased resistance is important for the treatment of inflammatory, allergic, and metabolic diseases. The aim of the present work was to evaluate the ability of violacein to change microbial patterns in the mammalian gut by favoring certain groups over the others in order to be used as a therapy for diseases associated with changes in the intestinal microflora. To do this, we used male Wistar rats, and administered violacein orally, in low (50 µg/ml) and high (500 µg/ml) doses for a month. Initially, the changes in the microbial diversity were observed by DGGE analyses that showed that the violacein significantly affects the gut microbiota of the rats. Pyrosequencing of 16S rDNA was then employed using a 454 GS Titanium platform, and the results demonstrated that higher taxonomic richness was observed with the low violacein treatment group, followed by the control group and high violacein treatment group. Modulation of the microbiota at the class level was observed in the low violacein dose, where Bacilli and Clostridia (Firmicutes) were found as dominant. For the high violacein dose, Bacilli followed by Clostridia and Actinobacteria were present as the major components. Further analyses are crucial for a better understanding of how violacein affects the gut microbiome and whether this change would be beneficial to the host, providing a framework for the development of alternative treatment strategies for intestinal diseases using this compound.

The Bisindole Alkaloid Caulerpin, from Seaweeds of the Genus Caulerpa, Attenuated Colon Damage in Murine Colitis Model.

Caulerpin (CLP), an alkaloid from algae of the genus Caulerpa, has shown anti-inflammatory activity. Therefore, this study aimed to analyze the effect of CLP in the murine model of peritonitis and ulcerative colitis. Firstly, the mice were submitted to peritonitis to evaluate which dose of CLP (40, 4, or 0.4 mg/kg) could decrease the inflammatory infiltration in the peritoneum. The most effective doses were 40 and 4 mg/kg. Then, C57BL/6 mice were submitted to colitis development with 3% dextran sulfate sodium (DSS) and treated with CLP at doses of 40 and 4 mg/kg. The disease development was analyzed through the disease activity index (DAI); furthermore, colonic tissue samples were submitted to histological analysis, NFκB determination, and in vitro culture for cytokines assay. Therefore, CLP at 4 mg/kg presented the best results, triggering improvement of DAI and attenuating the colon shortening and damage. This dose was able to reduce the TNF-α, IFN-γ, IL-6, IL-17, and NFκB p65 levels, and increased the levels of IL-10 in the colon tissue. Thus, CLP mice treatment at a dose of 4 mg/kg showed promising results in ameliorating the damage observed in the ulcerative colitis.

RNAm expression profile of cancer marker genes in HepG2 cells treated with different concentrations of a new indolin-3-one from Pseudomonas aeruginosa.

The present study tested the effects of a newly identified indolin-3-one compound (compound 1), produced by Pseudomonas aeruginosa, on HepG2 cells. The MTT assays demonstrated decreased metabolic activities in HepG2 cells treated with compound 1, with dose- and time-dependent intensifying effect, starting at a concentration of 40 µM. The IC50 after 24, 48, 72, and 96 h treatments were 41.35, 52.7, 92.79 and 66.65 µM of compound 1, respectively. Below 80 µM, no significative damage on erythrocytes membranes was observed by the hemolytic assays. The RT-qPCR revealed that the compound modulated key genes involved in carcinogenesis process, indicating possible indolin-3-one mechanisms of action. The data showed that gene expression alterations promoted by compound 1, in concentrations up to 60 µM after 48 h, led to a decrease in cellular progression and there was no direct cellular damage. In addition, non-cytotoxic concentrations of compound 1 halved the concentration of the chemotherapeutic doxorubicin, maintaining similar therapeutic effect against HepG2 cells. The novelty of the molecule and the biological activities observed in the present study emphasize the potential of the compound 1 in cancer therapy research.

Seasonality effect on the composition of oxindole alkaloids from distinct organs of Uncaria tomentosa from the Caribbean region of Costa Rica.

Uncaria tomentosa (Willd.) D.C. (Rubiaceae), commonly known as "Uña de Gato" or "Cat's Claw", is a tropical vine from the rainforest used in traditional medicine and spread through Central and South America, including Costa Rica. There is an increasing demand for medicinal extracts with biological activity attributed mainly to oxindole alkaloids (OA), where the ratio between tetracyclic (TOA) and pentacyclic oxindole alkaloids (POA) determines its feasibility for medicinal applications. The ratio is affected by distinct factors including the dynamics of environmental conditions during seasons. The purpose of the study was to assess the seasonality effect in oxindole alkaloids content in relation to plant organs from U. tomentosa grown in the Caribbean region of Costa Rica. Young leaves followed by mature leaves presented the highest amount of total OA during seasons; for these, isoryncophylline, pteropodine and isomitraphylline, were the predominant OA. The POA/TOA ratio of both leaf materials was nearly 1:1 (3.2 mg g-1: 3.1 mg g-1). Bark and root material showed a pentacyclic chemotype in all seasons with a ratio of 6:1 (6.7 mg g-1: 1.3 mg g-1) with pteropodine and isomitraphylline as the predominant POA. The POA content presented seasonality with a significant increase from rainy to dry season in young leaves, bark and roots. In contrast, TOA amount remained virtually unchanged in all plant parts. Humidity and temperature between the studied seasons were constant except for precipitation, reflecting that differences of water content had an effect in the POA amounts. Further studies of abiotic factors, like water stress, could explain the variation of POA content due to seasonality.

Effects of indirubin and isatin on cell viability, mutagenicity, genotoxicity and BAX/ERCC1 gene expression.

CONTEXT: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood. OBJECTIVE: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes. MATERIALS AND METHODS: HeLa and/or CHO-K1 cell lines were tested (3 or 24 h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72 h) tests after treatment with IRN (0.1 to 200 µM) or ISA (0.5 to 50 µM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24 h) by gavage (50, 100 and 150 mg/kg determined from the LD50 - 1 g/kg b.w.) and submitted to comet assay in vivo. RESULTS: IRN reduced the viability of CHO-K1 (24 h; 5 to 200 µM) and HeLa cells (10 to 200 µM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 µM; HeLa: 5 and 10 µM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24 h) at all doses tested. IRN (100 and 150 mg/kg) also induced genotoxicity in vivo (4 h). CONCLUSION: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.

Identification and evaluation of semiochemicals for the biological control of the beetle Omorgus suberosus (F.) (Coleoptera: Trogidae), a facultative predator of eggs of the sea turtle Lepidochelys olivacea (Eschscholtz).

The beetle Omorgus suberosus (F.) is a facultative predator of eggs of the olive ridley turtle Lepidochelys olivacea (Eschscholtz). Laboratory and field investigations were conducted in order to characterize volatile attractants of O. suberosus and to explore the potential for application of these volatiles in a selective mass trapping method. Headspace sorptive extraction (HSSE) coupled to thermo-desorption gas chromatography-mass spectrometry (TD-GC-MS) analysis of the volatile constituents from beetles or turtle nests revealed 24 potential compounds. However, electroantennographic (EAG) measurements revealed antennal sensitivity only to indole, linoleic acid, trimethylamine, dimethyl sulphide, dimethyl disulphide and ammonia. Behavioural tests showed that these compounds are highly attractive to O. suberosus. Field trapping experiments revealed that indole and ammonia were more attractive than the other volatile compounds and showed similar attractiveness to that produced by conventional baits (chicken feathers). The use of a combined bait of indole and NH3 would therefore be the most effective trap design. The data presented are the first to demonstrate effective massive capture of O. suberosus using an attractant-based trapping method. These findings have potential for the development of an efficient mass trapping method for control of this beetle as part of efforts towards conservation of L. olivacea at La Escobilla in Oaxaca, Mexico.

Multifunctional Monoamine Oxidases and Cholinesterases Inhibitory Effects, as well as UPLC-DAD-MS Chemical Profile of Alkaloid Fractions Obtained from Species of the Palicoureeae Tribe.

In the present study, the effects were evaluated of alkaloid fractions (AFs) from Psychotria species and correlated genera, Palicourea and Rudgea, on monoamine oxidases (MAOs) and cholinesterases (ChEs). By HPLC-DAD and UPLC-DAD-MS analyses, indole alkaloids (IA) were detected in all AFs. For the Psychotria and Palicourea species, these IA corresponded to tetrahydro-p-carboline alkaloids (THPCA). On the other hand, pyrrolidinoindoline core compounds were observed for Rudgea species. Regarding their pharmacological activities, none of the AFs was able to inhibit AChE. However, the BChE activity was impaired by the Psychotria and Palicourea AFs. In addition, MAO-A was inhibited by both AFs, but only Psychotria nemorosa AF was able to inhibit significantly MAO-B. Rudgea AFs demonstrated a poor inhibitory profile on MAO-A. Taken together, our results highlighted the Psychotria and Palicourea genera as important sources of scaffolds for the development of MAO-A and BChE inhibitors aiming at the treatment of neurodegenerative and neuropsychiatric diseases.

A new indole-alkaloid and a new phenolic-glycoside with cytotoxic activity from Strychnos fendleri.

A new indole alkaloid strychnosinol (1) and a new phenolic-glycoside (2) were isolated from the bark and leaves of Strychnos fendleri Sprague & Sandwith, together with six known compounds reported for the first time in this species. The structures of these compounds were determined on the basis of spectroscopic data; mainly those obtained by using (1)H and (13)C NMR (1D and 2D) and mass spectrometry. Strychnosinol (1) and the phenolic glycoside (2) together with compounds 3-8 were evaluated for cytotoxicity against a panel of five tumour cell lines; IC50 values between 0.090 and 0.227 µM for the human tumour cell lines were observed for compound 2.

Bioprospection of cytotoxic compounds in fungal strains recovered from sediments of the Brazilian coast.

The cytotoxic activities of extracts (50 µg/ml) from 48 fungal strains, recovered from sediments of Pecém's offshore port terminal (Northeast coast of Brazil), against HCT-116 colon cancer cell lines were investigated. The most promising extract was obtained from strain BRF082, identified as Dichotomomyces cejpii by phylogenetic analyses of partial RPB2 gene sequence. Thus, it was selected for bioassay-guided isolation of the cytotoxic compounds. Large-scale fermentation of BRF082 in potato dextrose broth, followed by chromatographic purification of the bioactive fractions from the liquid medium, yielded gliotoxin (4) and its derivatives acetylgliotoxin G (3), bis(dethio)bis(methylsulfanyl)gliotoxin (1), acetylgliotoxin (5), 6-acetylbis(dethio)bis(methylsulfanyl)gliotoxin (2), besides the quinazolinone alkaloid fiscalin B. All isolated compounds were tested for their cytotoxicities against the tumor cell lines HCT-116, revealing 4 and 3 as the most cytotoxic ones (IC50 0.41 and 1.06 µg/ml, resp.).

Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast.

A fungal strain of Aspergillus sp. (BRF 030) was isolated from the sediments collected in the northeast coast of Brazil, and the cytotoxic activity of its secondary metabolites was investigated against HCT-116 tumour cell line. The cytotoxicity-guided fractionation of the extracts from this fungus cultured in potato-dextrose-sea water for 14 days at room temperature yielded the hetero-spirocyclic γ-lactams pseurotin A (1), pseurotin D (2) and pseurotin FD-838 (7), the alkaloids fumitremorgin C (5), 12,13-dihydroxy fumitremorgin C (6), methylsulochrin (4) and bis(dethio)bis(methylthio)gliotoxin (3). Among them, fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6) were the most active. The cytotoxic activities of the extracts from Aspergillus sp. grown from 7 to 28 days were investigated, and they were associated with the kinetic production of the compounds. The most active extracts (14 and 21 days) were those with the highest relative concentrations of the compounds fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6).

Antimicrobial and allelopathic metabolites produced by Penicillium brasilianum.

Six known compounds, isoroquefortine C (1), griseofulvin (2), ergosterol peroxide (3), 3ß-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one (4), cerevisterol (5) and (22E,24R)-6ß-methoxyergosta-7,22-diene-3ß,5α-diol (6), were produced by the fungus Penicillium brasilianum, and their structures were elucidated by spectroscopic methods. This is the first report on isoroquefortine C as naturally occurring compound. Their bioactivities against five phytopathogenic fungi (Gibeberalla saubinetti, Fusarium solani, Botrytis cinerea, Colletotrichum gloeosporioides and Alternaria solani) and four pathogenic bacteria (Escherichia coli, Bacillus subtilis, Staphyloccocus aureus and Bacillus cereus), as well as allelopathic activities on Raphanus sativus were tested. Compound 1 exhibited a remarkable antifungal activity with minimum inhibitory concentration (MIC) of 12.5 µM against C. gloeosporioides, in comparison with positive control hymexazol (MIC 25 µM). Compound 2 displayed strong inhibitory effects on the growth of A. solani and S. aureus with MIC of 3.13 µM for each. Compounds 2 and 3 displayed a significant growth-inhibition activity on R. sativus.

Chemical composition of the bark of Tetrapterys mucronata and identification of acetylcholinesterase inhibitory constituents.

The secondary metabolite content of Tetrapterys mucronata, a poorly studied plant that is used occasionally in Brazil for the preparation of a psychotropic plant decoction called "Ayahuasca", was determined to establish its chemical composition and to search for acetylcholinesterase (AChE) inhibitors. The ethanolic extract of the bark of T. mucronata exhibited in vitro AChE inhibition in a TLC bioautography assay. To localize the active compounds, biological profiling for AChE inhibition was performed using at-line HPLC-microfractionation in 96-well plates and subsequent AChE inhibition bioautography. The analytical HPLC-PDA conditions were transferred geometrically to a preparative medium-pressure liquid chromatography column using chromatographic calculations for the efficient isolation of the active compounds at the milligram scale. Twenty-two compounds were isolated, of which six are new natural products. The structures of the new compounds (9, 10, 16-18, and 20) were elucidated by spectroscopic data interpretation. Compounds 1, 5, 6, 9, and 10 inhibited AChE with IC50 values below 15 µM.

A polymorphic form of 4,4-dimethyl-8-methylene-3-azabicyclo[3.3.1]non-2-en-2-yl 3-indolyl ketone, an indole alkaloid extracted from Aristotelia chilensis (maqui).

The title compound [systematic name: (4,4-dimethyl-8-methylene-3-azabicyclo[3.3.1]non-2-en-2-yl)(1H-indol-3-yl)methanone], C20H22N2O, (II), was obtained from mother liquors extracted from Aristotelia chilensis (commonly known as maqui), a native Chilean tree. The compound is a polymorphic form of that obtained from the same source and reported by Watson, Nagl, Silva, Cespedes & Jakupovic [Acta Cryst. (1989), C45, 1322-1324], (Ia). The molecule consists of an indolyl ketone fragment and a nested three-ring system, with both groups linked by a C-C bridge. Comparison of both forms shows that they do not differ in their gross features but in the relative orientation of the two ring systems, due to different rotations around the bridge, as measured by the O=C-C=N torsion angle [130.0 (7)° in (Ia) and 161.6 (2)° in (II)]. The resulting slight conformational differences are reflected in a number of intramolecular contacts being observed in (II) but not in (Ia). Regarding intermolecular interactions, both forms share a similar N-H···O synthon but with differing hydrogen-bonding strength, leading in both cases to C(6) catemers with different chain motifs. There are marked differences between the two forms regarding colour and the (de)localization of a double bond, which allows speculation about the possible existence of different variants of this type of molecule.

Indolo[3,2-a]carbazoles from a deep-water sponge of the genus Asteropus.

Two new indolo[3,2-a]carbazoles (1, 2) were isolated from a deep-water collection of a sponge of the genus Asteropus. The structures of 1 and 2 were determined through the analysis of spectroscopic data including mass spectrometry and 2D-NMR. Compound 1 showed minimum inhibitory concentrations of 25 µg/mL against the fungal pathogen Candida albicans and 50 µg/mL against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 1 and 2 showed no cytotoxicity against the PANC1 human pancreatic carcinoma and NCI/ADR-RES ovarian adenocarcinoma cell lines at our standard test concentration of 5 µg/mL.

Cat's claw oxindole alkaloid isomerization induced by cell incubation and cytotoxic activity against T24 and RT4 human bladder cancer cell lines.

The antitumor activity of Uncaria tomentosa, a native vine from the Amazonian rainforest, has been ascribed to pentacyclic oxindole alkaloids occurring in its bark. Former studies have shown that this activity, as well as its intensity, depends on whether cat's claw alkaloids occur as original compounds or isomerized derivatives. This work addresses this aspect, using T24 and RT4 human bladder cancer cell lines for that purpose. Bark samples were extracted by dynamic maceration, prepurified with cross-linked polyvinylpyrrolidone and properly fractioned by an ion exchange process to obtain an oxindole alkaloid purified fraction. Alkaloid isomerization was induced by heating it under reflux at 85 °C. Samples collected after 5, 15, and 45 min of heating were analyzed by HPLC-PDA, freeze-dried at once, and separately assayed using the non-isomerized purified fraction for comparison purposes. The latter showed significant and dose-dependent cytotoxic activity against both T24 and RT4 cancer cell lines (IC50: 164.13 and 137.23 µg/mL, respectively). However, results for both cell lines were equivalent to those observed for isomerized samples (p > 0.05). The alkaloid isomerization induced by the incubation conditions (buffered medium pH 7.4 and temperature 37 °C) helps to explain the similar results obtained from non-isomerized and isomerized samples. Mitraphylline, speciophylline, uncarine F, and, to a lesser degree, pteropodine were more susceptible to isomerization under the incubation conditions. Thus, the alkaloid profile of all fractions and their cytotoxic activities against T24 and RT4 human bladder cancer cell lines are determined to a large extent by the incubation conditions.