Croton stipulaceus Kunth, a native Mexican medicinal plant with antioxidant and anti-inflammatory activities / Croton stipulaceus Kunth, planta medicinal mexicana con actividad antioxidante y antiinflamatoria

Leaves of Croton stipulaceuswere extracted (EHex, ECHCl3and EEtOH extracts) to assesstheir antioxidant potential, anti-inflammatory activity in murine models and acute toxicity. EEtOH showed the highest effect in DPPH (37.80% inhibition), FRAP (1065.00 ± 55.30 µmolFe2+) and total polyphenols (231.24 ± 9.05 meq AG/gM). EHex was the most active, ~ 50% inhibition of TPA-induced ear edema; while EEtOH (dose of 2 mg/ear) showed the highest inhibition in the chronic model (97% inhibition), and inhibited MPO activity (48%). In carrageenan-induced edema, ECHCl3(dose 500 mg/kg) was the most active. None of the extracts showed acute toxicity (LD50) at 2 g/kg (p.o.). This work is the first report that supports the traditional use of C. stipulaceusas an anti-inflammatory.

De las hojas de Croton stipulaceusse obtuvieron diferentes extractos (EHex, ECHCl3y EEtOH) evaluando el potencial antioxidante y la actividad antiinflamatoria en modelos murinos y la toxicidad aguda. El EEtOH mostró mayor efecto en DPPH (37.80% inhibición), FRAP (1065.00 ± 55.30 µmolFe2+) y polifenolestotales (231.24 ± 9.05 meq AG/gM). El EHex fue el más activo, cercano al 50% de inhibición del edema auricular inducido con TPA; mientras que el EEtOH (dosis de 2 mg/oreja) mostró la mayor inhibición en el modelo crónico (97% inhibición), e inhibió la actividad de la MPO (48%). En el edema inducido con carragenina, el ECHCl3(dosis 500 mg/kg) fue el más activo. Ninguno de los extractos mostró una toxicidad aguda (DL50) mayor a 2 g/kg (p.o). Este trabajo es el primer reporte que sustenta el uso tradicional de C. stipulaceuscomo antiinflamatorio.

A chromosome-level genome assembly for Dracaena cochinchinensis reveals the molecular basis of its longevity and formation of dragon's blood.

Dracaena, a remarkably long-lived and slowly maturing species of plant, is world famous for its ability to produce dragon's blood, a precious traditional medicine used by different cultures since ancient times. However, there is no detailed and high-quality genome available for this species at present; thus, the molecular mechanisms that underlie its important traits are largely unknown. These factors seriously limit the protection and regeneration of this rare and endangered plant resource. Here, we sequenced and assembled the genome of Dracaena cochinchinensis at the chromosome level. The D. cochinchinensis genome covers 1.21 Gb with a scaffold N50 of 50.06 Mb and encodes 31 619 predicted protein-coding genes. Analysis showed that D. cochinchinensis has undergone two whole-genome duplications and two bursts of long terminal repeat insertions. The expansion of two gene classes, cis-zeatin O-glucosyltransferase and small auxin upregulated RNA, were found to account for its longevity and slow growth. Two transcription factors (bHLH and MYB) were found to be core regulators of the flavonoid biosynthesis pathway, and reactive oxygen species were identified as the specific signaling molecules responsible for the injury-induced formation of dragon's blood. Our study provides high-quality genomic information relating to D. cochinchinensis and significant insight into the molecular mechanisms responsible for its longevity and formation of dragon's blood. These findings will facilitate resource protection and sustainable utilization of Dracaena.

Evaluation of Antioxidant and Enzyme Inhibition Properties of Croton hirtus L'Hér. Extracts Obtained with Different Solvents.

Croton hirtus L'Hér methanol extract was studied by NMR and two different LC-DAD-MSn using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources to obtain a quali-quantitative fingerprint. Forty different phytochemicals were identified, and twenty of them were quantified, whereas the main constituents were dihydro α ionol-O-[arabinosil(1-6) glucoside] (133 mg/g), dihydro ß ionol-O-[arabinosil(1-6) glucoside] (80 mg/g), ß-sitosterol (49 mg/g), and isorhamnetin-3-O-rutinoside (26 mg/g). C. hirtus was extracted with different solvents-namely, water, methanol, dichloromethane, and ethyl acetate-and the extracts were assayed using different in vitro tests. The methanolic extracts presented the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) values. All the tested extracts exhibited inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with a higher activity observed for dichloromethane (AChE: 5.03 and BChE: 16.41 mgGALAE/g), while the methanolic extract showed highest impact against tyrosinase (49.83 mgKAE/g). Taken together, these findings suggest C. hirtus as a novel source of bioactive phytochemicals with potential for commercial development.

Novel findings to the biosynthetic pathway of magnoflorine and taspine through transcriptomic and metabolomic analysis of Croton draco (Euphorbiaceae).

BACKGROUND: Croton draco is an arboreal species and its latex as well as some other parts of the plant, are traditionally used in the treatment of a wide range of ailments and diseases. Alkaloids, such as magnoflorine, prevent early atherosclerosis progression while taspine, an abundant constituent of latex, has been described as a wound-healer and antitumor-agent. Despite the great interest for these and other secondary metabolites, no omics resources existed for the species and the biosynthetic pathways of these alkaloids remain largely unknown. RESULTS: To gain insights into the pathways involved in magnoflorine and taspine biosynthesis by C. draco and identify the key enzymes in these processes, we performed an integrated analysis of the transcriptome and metabolome in the major organs (roots, stem, leaves, inflorescences, and flowers) of this species. Transcript profiles were generated through high-throughput RNA-sequencing analysis while targeted and high resolution untargeted metabolomic profiling was also performed. The biosynthesis of these compounds appears to occur in the plant organs examined, but intermediaries may be translocated from the cells in which they are produced to other cells in which they accumulate. CONCLUSIONS: Our results provide a framework to better understand magnoflorine and taspine biosynthesis in C. draco. In addition, we demonstrate the potential of multi-omics approaches to identify candidate genes involved in the biosynthetic pathways of interest.

Biologically Active Orbitides from the Euphorbiaceae Family.

A comprehensive overview of natural orbitides isolated from Euphorbiaceae species and their most relevant biological activities are presented. Euphorbiaceae is a large and diverse family, which comprises about 300 genera, and is known as an important source of medicines and toxins. Several classes of secondary metabolites have been described for this taxon, however, orbitides have been broadly reported in Jatropha and Croton genera. Additionally, the latex is documented as the main source of orbitides in this family. Based on their structural and functional diversity, orbitides present a large variety of biological activities described as cytotoxicity, antimalarial, antibacterial, antifungal, enzymatic inhibition, and immunosuppressive, although the mechanism of action still needs to be further investigated. In recent years, the discovery of bioactive cyclic peptides from different sources has grown exponentially, making them promising molecules in the search for new drug leads. This review also highlights the attempts made by many researchers to organize the orbitides nomenclature and amino acid numbering, as well the important progress recently achieved in the biosynthetic study area.

The First Casbane Hydroperoxides EBC-304 and EBC-320 from the Australian Rainforest.

Investigation of the Australian rainforest plant Croton insularis led to isolation of the first casbane hydroperoxide diterpenes EBC-304 and EBC-320. Extensive DFT and electronic circular dichroism (ECD) calculations in combination with 2D NMR spectroscopy determined the absolute configurations. EBC-304 and EBC-320 both display significant cytotoxicity.

New clerodane diterpenoids from the twigs and leaves of Croton euryphyllus.

Three new nor-clerodane diterpenoids, crotoeurins A-C (1-3), together with four known ones were isolated from the twigs and leaves of Croton euryphyllus. Among them, crotoeurin A (1) is a new nor-clerodane diterpenoid dimer with a unique cyclobutane ring via a [2+2] cycloaddition. Their structures were elucidated by spectroscopic analyses and the stereochemistry of 1 was confirmed by single-crystal X-ray diffraction analysis. Compounds 1-3 exhibited neurite outgrowth-promoting activity on NGF-mediated PC12 cells at concentration of 10µM.

Ozone-induced responses in Croton floribundus Spreng. (Euphorbiaceae): metabolic cross-talk between volatile organic compounds and calcium oxalate crystal formation.

Here, we proposed that volatile organic compounds (VOC), specifically methyl salicylate (MeSA), mediate the formation of calcium oxalate crystals (COC) in the defence against ozone (O3) oxidative damage. We performed experiments using Croton floribundus, a pioneer tree species that is tolerant to O3 and widely distributed in the Brazilian forest. This species constitutively produces COC. We exposed plants to a controlled fumigation experiment and assessed biochemical, physiological, and morphological parameters. O3 induced a significant increase in the concentrations of constitutive oxygenated compounds, MeSA and terpenoids as well as in COC number. Our analysis supported the hypothesis that ozone-induced VOC (mainly MeSA) regulate ROS formation in a way that promotes the opening of calcium channels and the subsequent formation of COC in a fast and stable manner to stop the consequences of the reactive oxygen species in the tissue, indeed immobilising the excess calcium (caused by acute exposition to O3) that can be dangerous to the plant. To test this hypothesis, we performed an independent experiment spraying MeSA over C. floribundus plants and observed an increase in the number of COC, indicating that this compound has a potential to directly induce their formation. Thus, the tolerance of C. floribundus to O3 oxidative stress could be a consequence of a higher capacity for the production of VOC and COC rather than the modulation of antioxidant balance. We also present some insights into constitutive morphological features that may be related to the tolerance that this species exhibits to O3.

Efficiency of the Polyethylene-Glycol (PEG) Embedding Medium for Plant Histochemistry.

Histochemical analyses in plants are commonly performed on hand-made sections of fresh materials. The disadvantages of embedding in historesin, paraffin or paraplast® are the alterations to cellular contents, the high costs and few evident results, depending on the test. Polyethylene-glycol (PEG), as a low cost, hydrophilic medium that maintains most of the cellular features similar to fresh conditions, may be useful for obtaining good histochemical results in thinner and homogeneous sections. The current study aimed to compare the efficiency of PEG as an embedding medium for histochemical analyses of primary and secondary metabolites accumulation. Using hand-made sections of fresh samples (T1) as a comparison, we tested the influence of the use of Karnovsky's solution as a fixative (T2) versus embedding in PEG (T3). The samples herein analyzed comprise leaves, stems, seeds and insect galls of different plant species. Neither the Karnovsky's fixative nor the embedding in PEG altered the histochemical results for starch, lipids, terpenoids, proteins and reducing sugars in T1, T2, and T3. However, PEG binds to phenols, such as tannins, flavonoids and lignins, thereby presenting false negatives in T3.

Transcription profiles analysis of genes encoding 1-deoxy-D-xylulose 5-phosphate synthase and 2C-methyl-D-erythritol 4-phosphate synthase in plaunotol biosynthesis from Croton stellatopilosus.

Genes encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS; EC 2.2.1.7) and 2C-methyl-D-erythritol 4-phosphate synthase (MEPS; EC 1.1.1.267), the first two enzymes in the deoxyxylulose phosphate (DXP) pathway, were cloned from young leaves of Croton stellatopilosus, and designated as 1-deoxy-D-xylulose 5-phosphate synthase (CSDXS) and 2C-methyl-D-erythritol 4-phosphate synthase (CSMEPS), respectively. Analysis of deduced amino acid sequences of the CSDXS and the CSMEPS confirmed their nucleotide sequences as they shared high identities to other known DXSs and MEPSs in higher plants. Physiological roles of the CSDXS and the CSMEPS were determined for the mRNA expressions in leaves, twigs and roots. Transcription profiles analyses of the CSDXS and the CSMEPS genes were investigated using semi-quantitative RT-PCR technique. Relative intensities of the CSDXS and the CSMEPS expressions to house-keeping gene (18S rRNA) were calculated. The results indicated that the levels of mRNAs expressions of the CSDXS and the CSMEPS were high in leaves and twigs. This evidence was in line with the high content of plaunotol, accumulated in leaves and twigs. Neither the CSDXS nor the CSMEPS were expressed in roots, where plaunotol was not detected. From this study, it can be concluded that plaunotol is biosynthesized in the chloroplastic tissue and regulated by the CSDXS and the CSMEPS.

Establishment of Croton stellatopilosus suspension culture for geranylgeraniol production and diterpenoid biosynthesis.

Diterpenoids in higher plants are biosynthesized from isoprene units obtained from two distinct pathways: the mevalonate pathway and the deoxyxylulose phosphate pathway. The metabolic partitioning of both pathways in plant species is dependent upon the type of culture. In order to study the diterpenoid biosynthesis in Croton stellatopilosus cell culture, callus culture was firstly induced from C. stellatopilosus young leaves in Murashige and Skoog (MS) medium in the presence of 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. The suspension culture was further induced from its callus in the same medium without gelling agent. Detection of diterpenoid accumulation by gas chromatography-mass spectrometry revealed that a cell culture could accumulate a low amount of geranylgeraniol (GGOH) and a high content of fatty acids and phytosterols. To improve the GGOH production, the culture conditions were optimized by medium manipulation in terms of hormonal factors. The growth rates of cell cultures were similar in all kinds of media. The GGOH production curve indicated that GGOH plays an important role as a primary metabolite in the cell culture. The optimum medium for GGOH production was MS medium supplemented with 2.0 mg/l 2,4-D and 2 mg/l BA that could produce GGOH with a yield of 1.14 mg/g FW.

Biosynthesis of beta-sitosterol and stigmasterol in Croton sublyratus proceeds via a mixed origin of isoprene units.

A green callus culture of Croton sublyratus Kurz established from the leaf explants appeared to actively synthesize two well-known phytosterols, beta-sitosterol and stigmasterol. The phytosterol biosynthesis was highly active during the linear phase of the culture. Feeding of [1-13C]glucose into the callus culture at this growth phase showed that the label from glucose was highly incorporated into both phytosterols. Isolation of the labeled products followed by 13C NMR analysis revealed that the phytosterols had their 13C-labeling patterns consistent with the acquisition of isoprene units via both the mevalonate pathway and the deoxyxylulose pathway with relatively equal contribution. Since the biosynthesis of phytosterol has so far been reported to be mainly from the classical mevalonate pathway, this study provides a new evidence on the biosynthesis of phytosterols via the novel deoxyxylulose pathway.