On the effect of intramolecular H-bonding in the configurational assessment of polyhydroxylated compounds with computational methods. The hyacinthacines case.

Hyacinthacines are important members of the pyrrolizidine family, with several compounds having ambiguous, revised or unverified structures. Herein we thoroughly explored the performance DP4 and DP4+ for the in silico stereoassignment of hyacinthacines A1, A2 and five synthetic isomers. The results suggested that the quality of the predictions strongly depended on the conformational landscape provided by DFT energies, with five compounds correctly assigned. In the two cases incorrectly classified we found that the source of the problem was conformational in nature, with spurious conformations being considerably over-stabilized by intramolecular H-bondings. We showed that neglecting such shapes resulted in a noteworthy improvement, with all compounds correctly assigned in high confidence (>99.9%).

Prenatal exposure to integerrimine N-oxide enriched butanolic residue from Senecio brasiliensis affects behavior and striatal neurotransmitter levels of rats in adulthood.

Pyrrolizidine alkaloids (PAs) are toxins that are exclusively biosynthesized by plants and are commonly present in foods and herbs. PAs are usually associated with poisoning events in livestock and human beings. The aim of the present study was to evaluate the behavioral and neurochemical effects of prenatal exposure to PA integerrimine N-oxide of rats in adulthood. Pregnant Wistar rats received integerrimine N-oxide from the butanolic residue of Senecio brasiliensis by gavage on gestational days 6-20 at doses of 3, 6 and 9 mg/kg. During adulthood of the offspring, the following behavioral tests were performed: open-field, plus-maze, forced swimming, catalepsy and stereotypy. Histological analyses and monoamine levels were measured. Male offspring from dams that were exposed to 9 mg/kg showed an increase in locomotion in the open-field test, an increased frequency of entries and time spent in open arms in elevated plus-maze test, as well as decreased swimming time. In the female offspring from dams that were exposed to 9 mg/kg, there was an increased time of climbing in forced swimming and intensity of stereotyped behavior. The histological study indicates an increase in the number of multinucleated cells in the liver (6 and 9 mg/kg). In neurotransmitter analysis, specifically in the striatum, we observed change in dopamine and serotonin levels in the middle dose. Thus, our results indicate that prenatal exposure to integerrimine N-oxide changed behavior in adulthood and neurotransmitter levels in the striatum. Our results agree with previous studies, which showed that integerrimine N-oxide impaired physical and neurobehavioral development in childhood that can persist until adulthood.

Sesquiterpenes, flavonoids, shikimic acid derivatives and pyrrolizidine alkaloids from Senecio kingii Hook.

Twenty-four compounds including eleven eremophilanolides (1-11), one eremophilane (13), five shikimic acid derivatives (14-18), six flavonoids (19-24), and the macrocyclic unsaturated pyrrolizidine alkaloid integerrimine (25) were isolated from Senecio kingii, an endemic species from the Magallanes Region (Chile). Compounds 3, 5, 6, 8-11 and 13-18 have not been previously reported as natural products. Their molecular structures were determined by NMR spectroscopic analysis and comparison with published NMR data. An X-ray-analysis of compound 3 has been performed. Their insecticidal and antifungal activities were tested, being compound 3 the strongest insect antifeedant. Compounds 6, 9 and 18 were moderate antifungals.

Complete 1H NMR assignments of pyrrolizidine alkaloids and a new eudesmanoid from Senecio polypodioides.

Chemical investigation of the aerial parts of Senecio polypodioides lead to the isolation of the new eudesmanoid 1ß-angeloyloxyeudesm-7-ene-4ß,9α-diol (1) and the known dirhamnosyl flavonoid lespidin (3), while from roots, the known 7ß-angeloyloxy-1-methylene-8α-pyrrolizidine (5) and sarracine N-oxide (6), as well as the new neosarracine N-oxide (8), were obtained. The structure of 1 and 8 was elucidated by spectral means. Complete assignments of the (1)H NMR data for 5, 6, sarracine (7), and 8 were made using one-dimensional and two-dimensional NMR experiments and by application of the iterative full spin analysis of the PERCH NMR software.

The selective cytotoxicity elicited by phytochemical extract from Senecio graveolens (Asteraceae) on breast cancer cells is enhanced by hypoxia.

Breast cancer is the second cause of cancer­related deaths in woman and the incidence of the disease has increased worldwide, in part due to improvements in early detection. Several drugs with anticancer effects have been extracted from plants in the last 20 years, many of which are particularly effective against breast cancer cells. In particular, we have become interested in the ethanolic extract from Senecio graveolens (synonym of S. nutans), a plant commonly called Chachacoma, in an effort to isolate compounds that could demonstrate cytotoxic effects on breast cancer cells. Senecio (Asteraceae) is the largest gender in Chile comprising approximatly 200 species. These herbs inhabit areas over 3,500 meters above the sea level in the Andes Mountains. S. graveolens is commonly used by local communities for its medicinal properties, particularly its capacity to ameliorate high-altitude-associated sickness. The cytotoxic effect of the alcoholic extract from S. graveolens, as well as its most abundant compound 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone, were tested in the breast cancer cell lines ZR-75-1, MCF-7 and MDA-MB­231, and non-tumorigenic MCF-10F cells. We show that the phytochemical extract was able to induce cytotoxicity in cancer cells but not in MCF-10F. Importantly, this effect was enhanced under hypoxic conditions. However, 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone, the main compound, did not by itself show an effective anticarcinogenic activity in comparison to the whole extract. Interestingly, the cytotoxic effect of the phytochemical extract was dependent on the basal MnSOD protein expression. Thus, cytotoxicity was increased when MnSOD levels were low, but resistance was evident when protein levels were high. Additionally, the crude extract seems to trigger cell death by a variety of processes, including autophagy, apoptosis and necrosis, in MCF-7 cells. In summary, S. graveolens extract possess anticancer activity displaying a specific cytotoxic effect on cancer cells, thus serving as a potential source of phytochemical compounds for cancer treatment.

Structural elucidation and NMR assignments of a new pyrrolizidine alkaloid from Crotalaria vitellina Ker Gawl.

A new pyrrolizidine alkaloid, named crotavitelin, was isolated from fruits of Crotalaria vitellina, Fabaceae (Papilionoideae). The structure was established by spectroscopic techniques such as one-dimensional and two-dimensional NMR, IR, and MS.

Supinidine viridiflorates from the roots of Chromolaena pulchella.

The alkaloid extract from the roots of Chromolaena pulchella provided two new pyrrolizidine alkaloids, elucidated as (-)-supinidine triviridiflorate (1) and (-)-supinidine diviridiflorate (2) based on their physical and spectroscopic properties. Their absolute configuration was determined by chemical correlation with (-)-supinidine (3) and (+)-viridifloric acid (4).

Pyrrolizidine alkaloids of the endemic Mexican genus Pittocaulon and assignment of stereoisomeric 1,2-saturated necine bases.

The endemic Mexican genus Pittocaulon (subtribe Tussilagininae, tribe Senecioneae, Asteraceae) belongs to a monophyletic group of genera distributed in Mexico and North America. The five Pittocaulon species represent shrubs with broom-like succulent branches. All species were found to contain pyrrolizidine alkaloids (PAs). With one exception (i.e., stems of Pittocaulon velatum are devoid of PAs) PAs were found in all plant organs with the highest levels (up to 0.3% of dry weight) in the flower heads. Three structural types of PAs were found: (1) macrocyclic otonecine esters, e.g. senkirkine and acetylpetasitenine; (2) macrocyclic retronecine esters, e.g. senecionine, only found in roots, and (3) monoesters of 1,2-saturated necines with angelic acid. For an unambiguous assignment of the different stereoisomeric 1,2-saturated necine bases a GC-MS method was established that allows the separation and identification of the four stereoisomers as their diacetyl or trimethylsilyl derivatives. All otonecine esters that generally do not form N-oxides and the 1,2-saturated PAs were exclusively found as free bases, while the 1,2-unsaturated 7-angeloylheliotridine occurring in P. velatum was found only as its N-oxide. In a comparative study the 1H and 13C NMR spectra of the four stereoisomeric necine bases were completely assigned by the use of DEPT-135, H,H-COSY, H,C-HSQC and H,H-NOESY experiments and by iterative analysis of the 1H NMR spectra. Based on these methods the PA monoesters occurring in Pittocaulon praecox and P. velatum were assigned as 7-O-angeloyl ester respectively 9-O-angeloyl ester of dihydroxyheliotridane which could be identified for the first time as naturally occurring necine base. Unexpectedly, in the monoesters isolated from the three other Pittocaulon species dihydroxyheliotridane is replaced by the necine base turneforcidine with opposite configuration at C-1 and C-7. The species-specific and organ-typical PA profiles of the five Pittocaulon species are discussed in a biogenetic context.

Preventive activity of pyrrolizidine alkaloids from Seneciobrasiliensis (Asteraceae) on gastric and duodenal induced ulcer on mice and rats.

The alkaloid extract of Senecio brasiliensis inflorescences contain a mixture of the pyrrolizidine alkaloids (PA) senecionine, integerrimine, retrorsine, usaramine and seneciphylline. We evaluated this PA mixture on preventive antiulcerogenic effects on standard rodent models of induced gastric and duodenal ulcers. In the HCl/ethanol, indomethacin-bethanechol and hypothermic-restraint-induced gastric ulcer, the lesion was significantly inhibited by PA (p.o.) (p < 0.001). In the pylorus-ligature, PA (i.d.), significantly increased the gastric juice content and the pH values and decreased the acid output. In the cysteamine induced duodenal ulcers, PA (p.o.) showed significant inhibition (p < 0.001) of the duodenal lesions when compared to the respective control. The levels of the somatostatin hormone in the blood samples of animals pre-treated with the PA (12.5 mg/kg) and the free mucus and prostaglandin synthesis also increased (p < 0.001) after administration of PA extract (p.o.). The results suggested that the PA extract from Senecio brasiliensis inflorescences presents a significant anti-ulcer effect in the selected ulcer models. The mechanism involved with the action of the PA extract is the cytoprotection. Additional studies are in progress to determine other possible mechanisms involved with effect of the PA as anti-ulcer agents.

Modulation of gastrin and epidermal growth factor by pyrrolizidine alkaloids obtained from Senecio brasiliensis in acute and chronic induced gastric ulcers.

We investigated the antiulcerogenic activity of pyrrolizidine alkaloids (PAs) integerrimine, retrorsine, senecionine, usaramine and seneciplhylline, an alkaloidal extract obtained from Senecio brasiliensis. The PA extract demonstrated significantly activity in both, acute and chronic gastric ulcers on rats. The effects of PA extract were dose dependent. The mechanisms implicated on this activity were evaluated by determination of gastrin plasma levels in rats subjected to the acute treatment with PA extract and by expression of mRNA of Epidermal Growth Factor (EGF) after chronic treatment with this extract. The results showed that the PA extract increased both the levels of gastrin and the expression of EGF on these animals. Moreover, the histological examinations showed a reduction of exfoliation of superficial cells, hemorrhages and blood cell infiltration. We concluded that the PAs showed an important and qualitative antiulcerogenic activity mediated by increase in gastrin secretion and mRNA expression of EGF.

Synthesis of (1R,2R,7R,7aS)-1-hydroxymethyl-2,3,5,6,7,7a-hexahydropyrrolizidine-1,2,7-triol from rosmarinine degradation.

A novel tetrahydroxypyrrolizidine alkaloid (6) was obtained from rosmarinine degradation in 23% yield (isolated from Senecio callosas).

Structure-activity relationships of pyrrolizidine alkaloids in insect chemical defense against the orb-weaving spider Nephila clavipes.

Pyrrolizidine alkaloids (PAs) are known to protect Arctiidae moths and Danainae and Ithomiinae butterflies against the orb-weaving spider Nephila clavipes (Araneae, Araneidae, Tetragnathinae), which liberates adults of these insects unharmed from its web. We tested against this spider the role of eight PAs and one derived structure [an 89:11 mixture of the 12-membered macrocyclic diester free base integerrimine and senecionine and the respective N-oxide; two hydrolysis products from this mixture (the necine base retronecine, its respective N-oxide, and a mixture of integerriminic and senecionic necic acids); the 12-membered macrocyclic senkirkine; the 9-O-monoester free base senecioylretronecine and its respective N-oxide; and the 9-O-monoester free base callimorphine (a PA biosynthesized only by insects from retronecine)]. The mixture integerrimine-senecionine N-oxide seems to be more active than the respective free base [LibD50 (liberation dose 50) = 0.042 and 0.153 microg/dry weight of prey, respectively], but the difference in activity between the N-oxide and free base of the 9-O-monoester senecioylretronecine was slight (LibD50 = 0.167 and 0.104, respectively). Senkirkine, an otonecine base PA that does not form N-oxide and is not found in insects, was the less active, showing the highest LibD50 (0.354). The difference in antipredator activity between N-oxides and free bases from macrocyclic diesters and monoesters may be correlated with physicochemical properties of these molecules in interaction with the Nephila receptors. For the active structures, there was a significant correlation between dosage and antipredator activity. Both forms of retronecine and a necic acid mixture were inactive, supporting the hypothesis that PAs biosynthesized by insects from retronecine were originally produced and stored in order to optimize chemical defense. Comparison of dose/activity data with reported amounts of PAs in butterflies suggested that, in general, PA-specialist insects are protected against predation by Nephila.

Defensive chemistry of Senecio miser.

Three eremophilanolides, 1alpha-acetoxy-8beta-methoxy-10betaH-eremophil-7(11)-en-8alpha,12-olide (1); 1alpha-angeloyloxy-6beta-hydroxy-8beta-methoxy-10betaH-eremophil-7(11)-en-8alpha,12-olide (2); and 1alpha-angeloyloxy-8betaH,10betaH-eremophil-7(11)-en-8alpha,12-olide (3), and two pyrrolizidine alkaloids, integerrimine (4) and its N-oxide (5), were isolated from bioactive fractions of Senecio miser. The structures of the new compounds 1 and 2 were established by NMR spectroscopic analysis and chemical transformation. The X-ray analysis of compound 1 was also performed. Eremophilanolides 1 and 2 and alkaloids 4 and 5 were found to be strong insect antifeedants, further supporting a proposed defensive role for these classes of compounds.

Pyrrolizidine alkaloids: a word of caution

Most plant bearing pyrrolizidine alkaloids (PAs) are toxic to men and animals. These natural products are recognized to be hepatotoxic, pneumotoxic, carcinogenic and mutagenic. Thus, the presence of toxic pyrrolizidine alkaloids in certain medicinal plants entails a serious health risk. Moreover, people are exposed to undetermined toxicity hazards of pyrrolizidine alkaloid-containing plants due to the consumption of milk and meat from chronically PA-poisoned animals and honey from wild bees. Symphytum officinale (comfrey) has been used freely as tea, topical cream, salad and beverage. This is a clear and actual example of how lack of knowledge about chemistry and toxicology of a plant made easy its spreading in the consumer society as beneficial and safe.

Hepatotoxic pyrrolizidine alkaloids in the Mexican medicinal plant Packera candidissima (Asteraceae: Senecioneae).

A study of the hepatotoxic pyrrolizidine alkaloids (PAs) contained in Packera candidissima (Greene) Weber & Löve (Senecio candidissimus Greene), a Mexican medicinal plant used for the treatment of kidney ailments and noted for its antiseptic properties, is reported. Analysis by TLC and GC-MS have shown the presence in high levels of both PAs and their N-oxides in the root (0.76% dry weight) and the aerial parts (0.36% dry weight) of the plant material. GC-MS analysis showed the presence of senecionine, integerrimine, retrorsine, and usaramine in the aerial parts. The root was found to contain senkirkine as the major component. These results indicate that users of this herb are at high risk of poisoning, especially members of certain cultural groups in Northern Mexico and the Hispanic population in the southwestern United States. In addition, two sesquiterpenes, neoadenostylone and epineoadenostylone were identified from the neutral extracts of this plant material. The 6 alpha-angeloyloxy-9-oxo-delta 10(1)-furanoeremophilane is reported for the first time as a natural product.

Genotoxic action of an aqueous extract of Heliotropium curassavicum var. argentinum.

Heliotropium curassavicum var. argentinum is widely employed in gout, rheumatism, neuralgias, arteriosclerotic disorders, muscular algias, phlebitis, varix and other illnesses. In order to analyze the genotoxic effect produced in vitro by this medicinal plant, chromosomal aberrations (CA), mitotic index (MI) and anaphase delay (AD) were studied in the CHO cell line, with and without the addition of S9 mix. Prepared according to the Argentine pharmacopeia 0.1, 1, 10 and 100 micrograms/ml plant decoction (aqueous extract) were assayed. One hundred cells per culture were studied for CA and AD, while MI was calculated for 2000 nuclei. The results revealed a significant increase in the percentage of abnormal metaphases (p less than 0.001) and in total aberrations (p less than 0.001). Both the MI and the AD affected the cell cycle. All results were enhanced by the addition of an S9 fraction. The toxic effect could be associated with pyrrolizidine alkaloids and their N-oxides, which through a process of in vitro metabolism become activated by microsomal oxidation and change into pyrrolic derivatives.