Antifungal and Antibiofilm Activity of Riparin III against Dermatophytes.

The ability of dermatophytes to develop biofilms is possibly involved in therapeutic failure because biofilms impair drug effectiveness in the infected tissues. Research to find new drugs with antibiofilm activity against dermatophytes is crucial. In this way, riparins, a class of alkaloids that contain an amide group, are promising antifungal compounds. In this study, we evaluated the antifungal and antibiofilm activity of riparin III (RIP3) against Trichophyton rubrum, Microsporum canis, and Nannizzia gypsea strains. We used ciclopirox (CPX) as a positive control. The effects of RIP3 on fungal growth were evaluated by the microdilution technique. The quantification of the biofilm biomass in vitro was assessed by crystal violet, and the biofilm viability was assessed by quantifying the CFU number. The ex vivo model was performed on human nail fragments, which were evaluated by visualization under light microscopy and by quantifying the CFU number (viability). Finally, we evaluated whether RIP3 inhibits sulfite production in T. rubrum. RIP3 inhibited the growth of T. rubrum and M. canis from 128 mg/L and N. gypsea from 256 mg/L. The results showed that RIP3 is a fungicide. Regarding antibiofilm activity, RIP3 inhibited biofilm formation and viability in vitro and ex vivo. Moreover, RIP3 inhibited the secretion of sulfite significantly and was more potent than CPX. In conclusion, the results indicate that RIP3 is a promising antifungal agent against biofilms of dermatophytes and might inhibit sulfite secretion, one relevant virulence factor.

New ent-kaurene-type nor-diterpene and other compounds isolated from Annona vepretorum Mart. (Annonaceae).

A new nor-ent-kaurene diterpene and ten other compounds were isolated from Annona vepretorum stems, including four kaurene diterpenes, three alkamides, one sesquiterpene and two steroids. Their chemical structures were elucidated using spectroscopic methods, including 1D-, 2D-NMR, and HRESIMS. The absolute configuration of compounds 1, 5, 8, 9 and 10 was confirmed by CD experiments. Compounds 1-5 and 8-10 were evaluated for cytotoxic activity using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT method, against three human carcinoma cell lines: human colon (HCT-116), glioblastoma (SF295) and prostate (PC3). However, all isolated compounds exhibited low cytotoxic activity.

Alkylamides from Acmella oleracea: antinociceptive effect and molecular docking with cannabinoid and TRPV1 receptors.

Alkylamides are secondary metabolites in Acmella oleracea and display wide applications in treating several diseases. Since alkylamides can inhibit pain, this work aims to evaluate the antinociceptive profile of A. Oleracea methanolic extracts used in vivo and in silico assays. The extracts inhibited the neurogenic and inflammatory phases of the formalin test, ratifying the antinociceptive effect of alkylamides. Furthermore, the results from molecular docking demonstrated the interaction of A. oleracea alkylamides with the CB1/CB2 and TRPV1 receptors. Additionally, the crude methanolic extract of flowers did not induce potential side effects related to the classical cannabinoid tetrad: hypolocomotion and catalepsy. In conclusion, this work confirms the potential of the alkylamides of A. Oleracea as antinociceptive agents and, for the first time, correlates its effects with the endocannabinoid and vanilloid systems through in silico assays.

Solanum lycopersicum Seedlings. Metabolic Responses Induced by the Alkamide Affinin.

Alkamides have been observed to interact in different ways in several superior organisms and have been used in traditional medicine in many countries e.g., to relieve pain. Previous studies showed that affinin when applied to other plant species induces prominent changes in the root architecture and induces transcriptional adjustments; however, little is known about the metabolic pathways recruited by plants in response to alkamides. Previous published work with Arabidopsis seedlings treated in vitro with affinin at 50 µM significantly reduced primary root length. In tomato seedlings, that concentration did not reduce root growth but increase the number and length of lateral roots. Non-targeted metabolomic analysis by Gas Chromatography couplet to Mass Spectrometry (GC/EIMS) showed that, in tomato seedlings, affinin increased the accumulation of several metabolites leading to an enrichment of several metabolic pathways. Affinin at 100 µM alters the accumulation of metabolites such as organic acids, amino acids, sugars, and fatty acids. Finally, our results showed a response possibly associated with nitrogen, GABA shunt and serine pathways, in addition to a possible alteration in the mitochondrial electron transport chain (ETC), interesting topics to understand the molecular and metabolic mechanisms in response to alkamide in plants.

Global gene expression analyses of the alkamide-producing plant Heliopsis longipes supports a polyketide synthase-mediated biosynthesis pathway.

BACKGROUND: Alkamides are plant-specific bioactive molecules. They are low molecular weight N-substituted α-unsaturated acyl amides that display biological explicit activities in different organisms from bacteria, fungi, insects to mammals and plants. The acyl chain has been proposed to be biosynthesized from a fatty acid; however, this has not been demonstrated yet. Heliopsis longipes (Asteraceae) accumulates in root a C10 alkamide called affinin in its roots, but not in leaves. The closely related species Heliopsis annua does not produce alkamides. To elucidate the biosynthetic pathway of the alkamides acyl chain, a comparative global gene expression analysis contrasting roots and leaves of both species was performed. METHODS: Transcriptomics analysis allowed to identify genes highly expressed in H. longipes roots, but not in tissues and species that do not accumulate alkamides. The first domain searched was the Ketosynthase (KS) domain. The phylogenetic analysis using sequences of the KS domain of FAS and PKS from different organisms, revealed that KS domains of the differentially expressed transcripts in H. longipes roots and the KS domain found in transcripts of Echinacea purpurea, another alkamides producer species, were grouped together with a high bootstrap value of 100%, sharing great similarity. Among the annotated transcripts, we found some coding for the enzymatic domains KS, AT, ACP, DH, OR and TE, which presented higher expression in H. longipes roots than in leaves. The expression level of these genes was further evaluated by qRT-PCR. All unigenes tested showed higher expression in H. longipes roots than in any the other samples. Based on this and considering that the acyl chain of affinin presents unsaturated bonds at even C numbers, we propose a new putative biosynthesis pathway mediated by a four modules polyketide synthase (PKS). RESULTS: The global gene expression analysis led to the selection of a set of candidate genes involved in the biosynthesis of the acyl chain of affinin, suggesting that it may be performed by a non-iterative, partially reductive, four module type I PKS complex (PKS alk) previously thought to be absent from the plant kingdom.

Affinin and hexahydroaffinin: Chemistry and toxicological profile.

The present work aimed to determine the safety parameters of two new alkamides, affinin and hexahydroaffinin, with antinociceptive activity. To predict the preliminary acute toxicity, we used the acute and subchronic toxicity (50 mg/kg, orally [po]) in Swiss Webster mice. Genotoxicity assayed via analysis of cell micronuclei of the femoral bone marrow in mice; at the same time, metabolic parameters determined from peripheral blood samples. Furthermore, to discard the neuropharmacological effects, we assessed the ambulatory activity in mice to determine the possible effects in the central nervous system. Finally, we used capsaicin as a positive control of alkamides. According to our results, hexahydroaffinin (LD50 ≥ 5,000 mg/kg, po) is significantly less noxious than affinin (LD50 = 1,442.2 mg/kg, po) or capsaicin (LD50 = 489.9 mg/kg, po). In subchronic administration, we did not observe any changes in hematological or biochemical parameters in any compound analyzed from peripheral blood samples. Finally, the data from the genotoxicity assay showed micronuclei formation in 28%, 5%, and 3% of mice in the capsaicin, affinin, and hexahydroaffinin groups, respectively. With the results obtained in the present investigation, we suggest that affinin and hexahydroaffinin are not only useful candidates for possible new drugs but also safe compounds.

Glucosinolate catabolism during postharvest drying determines the ratio of bioactive macamides to deaminated benzenoids in Lepidium meyenii (maca) root flour.

Postharvest processing of maca (Lepidium meyenii Walp., Brassicaceae), a traditional high-altitude Andean root crop, involves slow field drying prior to milling into flour. The progressive tissue dehydration and release of hydrolytic enzymes and substrates from cellular compartments results in the slow accumulation of free monosaccharides, fatty acids and amino acids. A more complex, and faster, kinetic profile is that of glucosinolate breakdown. A number of reactive transient and stable accumulation products are generated during drying, some of which have noteworthy bioactive properties. Among these are macamides, inhibitors of endocannabinoid neurotransmitter degradation in mammalian nervous systems. They result from the condensation of benzyl amine, a glucosinolate hydrolysis product, with free fatty acids released from lipid hydrolysis. Recent research has focused on developing drying processes under controlled conditions that can modulate the biochemistry of glucosinolate hydrolysis to optimize the content of bioactive compounds in the root flour. Low temperature (35 °C) oven-drying of shredded maca roots under controlled air flow generates benzyl amine as primary accumulation product, accounting for up to 94% of hydrolyzed glucosinolate in the flour. Kinetic evidence suggests that both deaminated benzenoids and macamides are allocated from the benzylamine pool through amine oxidase activity or condensation with free fatty acids, accounting for the remaining hydrolyzed glucosinolate (<5%). These activities determine the allocation to either one of these pathways. Later stages of dehydration result in shifts in the molar ratios of deaminated benzenoids, the accumulation of benzoic acid esters and benzyl alcohol. We propose that these are the result of changes in the rates of the reductive and oxidative half-reactions of endogenous aldehyde dehydrogenases. It is the ratio of benzylamine deamination to amide formation that determines the eventual yields of macamides in relation to benzenoids and their esters in maca flour.

1 H and 13 C NMR data, occurrence, biosynthesis, and biological activity of Piper amides.

Alkamides are the major and characteristic chemical compounds of the plants belonging to the Piper genus. These compounds are responsible for the flavor of pepper spices and for its broad use in cuisine across many regions of the world. Humans are in contact every day with these substances, which additionally show a broad variety of pharmacological activities, making them an important research target. A large amount of NMR data for these natural products is dispersed throughout literature. Its organization will help those research groups interested in their identification and structural elucidation. This review summarizes the 1 H and 13 C NMR data of 268 Piper amides in a systematic and orderly way, with a discussion on their biological activities, biosynthetic aspects, and NMR analysis of typical and relevant aspects of this information.

Is the hype around the reproductive health claims of maca (Lepidium meyenii Walp.) justified?

ETHNOPHARMACOLOGICAL RELEVANCE: Maca - Lepidium meyenii Walp. has been cultivated and used by Andean people for over 1300-2000 years in Peru as food and medicine. Starting in the late 1990's it has developed into an important herbal medicine in China and is now cultivated there widely, too AIM OF STUDY: This study aims to provide an insight into the emergence of maca on the global market as an alternative remedy to treat reproductive health related problems in both men and women and to critically assess these health claims. METHODOLOGY: A search of electronic databases such as EMBASE and a hand-search was done to acquire peer-reviewed articles and reports about maca. RESULTS AND DISCUSSION: Lepidium meyenii is used traditionally as a tonic, fertility enhancer for both humans and cattle, and to treat a variety of ailments such as rheumatism, respiratory disorders and anaemia among others. Maca root is cooked, baked, fermented as a drink and made into porridge. In the last twenty years, maca was introduced onto the global market and demand has dramatically grown over this time with its promotion on the internet, as the 'Peruvian Ginseng' for libido and fertility enhancement. It has also been said to treat menopausal symptoms, erectile dysfunction and benign prostatic hyperplasia. The sky-rocketing demand for the plant has seen a shift from traditional cultivation methods to mass production practices with the use of fertilisers and also pesticides; as maca is now grown in areas other than the Andes such as in the Yunnan province in China. This can potentially affect the phytochemistry and composition of the plant and thus, the quality, safety and efficacy of maca products. Meanwhile, research into maca's medicinal properties has followed the spike in popularity of maca and has been focused mainly on maca's aphrodisiac and fertility enhancing properties. So far, the in vivo studies and clinical trials conducted have yielded inconclusive results. Some of the key limitations reside in methodology and sample size. Chemical profiling, led to the discovery of new compounds unique to maca, such as, 'macamides' and also other active metabolites like the glucosinolates; to which the medicinal effects of maca have been ascribed but cannot be confirmed due to lack of data. CONCLUSIONS: To date, the health claims of maca cannot be fully supported from a scientific standpoint and more research is needed. It appears that the indigenous local knowledge about the health benefits of maca has been dragged out of context to fit the demands of a growing market for herbal remedies. This globalisation (or hype esp. in China) also has had serious consequences for the local producers in Peru. The lack of protocols to regulate the production and marketing of maca during this rapid expansion, poses a threat to both the safety of consumers and the sustainability of supply.

Secondary Metabolic Profiles of Two Cultivars of Piper nigrum (Black Pepper) Resulting from Infection by Fusarium solani f. sp. piperis.

Bragantina and Cingapura are the main black pepper (Piper nigrum L.) cultivars and the Pará state is the largest producer in Brazil with about 90% of national production, representing the third largest production in the world. The infection of Fusarium solani f. sp. piperis, the causal agent of Fusarium disease in black pepper, was monitored on the cultivars Bragantina (susceptible) and Cingapura (tolerant), during 45 days' post infection (dpi). Gas Chromatography-Mass spectrometry (GC-MS) analysis of the volatile concentrates of both cultivars showed that the Bragantina responded with the production of higher contents of α-bisabolol at 21 dpi and a decrease of elemol, mostly at 30 dpi; while Cingapura displayed an decrease of δ-elemene production, except at 15 dpi. The phenolic content determined by the Folin Ciocalteu method showed an increase in the leaves of plants inoculated at 7 dpi (Bragantina) and 7-15 dpi (Cingapura); in the roots, the infection caused a phenolic content decrease in Bragantina cultivar at 45 dpi and an increase in the Cingapura cultivar at 15, 30 and 45 dpi. High Performance Liquid Chromatography-Mass spectrometry (HPLC-MS) analysis of the root extracts showed a qualitative variation of alkamides during infection. The results indicated that there is a possible relationship between secondary metabolites and tolerance against phytopathogens.

Spilanthol: occurrence, extraction, chemistry and biological activities

Abstract Spilanthol (C14H23NO, 221.339 g/mol) is a bioactive compound that is found in many different plants that are used as traditional remedies throughout the world. It is present in Heliopsis longipes and several species in the genus Acmella, including A. oleracea L., also known as paracress and jambu. Its leaves and flowers have sensory properties (pungency, tingling, numbing, mouth-watering) that make it a popular spice and ingredient in several Brazilian dishes. Spilanthol can exert a variety of biological and pharmacological effects including analgesic, neuroprotective, antioxidant, antimutagenic, anti-cancer, anti-inflammatory, antimicrobial, antilarvicidal and insecticidal activities. So, the aim of this review is to present a literature review on the spilanthol that describes its occurrence, chemistry, extraction and biological activities.

Bioactive maca (Lepidium meyenii) alkamides are a result of traditional Andean postharvest drying practices.

Maca, Lepidium meyenii Walpers (Brassicaceae), is an annual herbaceous plant native to the high plateaus of the Peruvian central Andes. Its underground storage hypocotyls have been a traditional medicinal agent and dietary staple since pre-Columbian times. Reported properties include energizing and fertility-enhancing effects. Published reports have focused on the benzylalkamides (macamides) present in dry hypocotyls as one of the main bioactive components. Macamides are secondary amides formed by benzylamine and a fatty acid moiety, with varying hydrocarbon chain lengths and degree of unsaturation. Although it has been assumed that they are usually present in fresh undamaged tissues, analyses show them to be essentially absent from them. However, hypocotyls dried by traditional Andean postharvest practices or industrial oven drying contain up to 800µgg(-1) dry wt (2.3µmolg(-1) dry wt) of macamides. In this study, the generation of macamides and their putative precursors were studied during nine-week traditional drying trials at 4200m altitude and in ovens under laboratory conditions. Freeze-thaw cycles in the open field during drying result in tissue maceration and release of free fatty acids from storage and membrane lipids up to levels of 1200µgg(-1) dry wt (4.3µmolg(-1) dry wt). Endogenous metabolism of the isothiocyanates generated from glucosinolate hydrolysis during drying results in maximal benzylamine values of 4300µgg(-1) dry wt (40.2µmolg(-1) dry wt). Pearson correlation coefficients of the accumulation profiles of benzylamine and free fatty acid to that of macamides showed good values of 0.898 and 0.934, respectively, suggesting that both provide sufficient substrate for amide synthesis during the drying process.

Evaluation of the anti-inflammatory activity of riparin II (O-methil-N-2-hidroxi-benzoyl tyramine) in animal models.

Riparin II (RipII), an alkamide isolated from the green fruit of Aniba riparia, was tested in the various animal models of inflammation to investigate its anti-inflammatory activity. Male Wistar rats (180-240g) were treated with RipII by gavage at doses 25 or 50mg/kg, before initiating the inflammatory responses. The tests used were paw edema induced by carrageenan, dextran, histamine or serotonin; peritonitis induced by carrageenan and fMLP, as well as the measurement of MPO activity, TNF-α and Il-1ß amount in the peritoneal fluid. In the animal models of carrageenan and dextran-induced paw edema, the animals treated with RipII showed lower edema than those of the control group. Treatment with RipII also reduced the paw edema induced by histamine but not serotonin. In the carrageenan-induced peritonitis model, treatment with RipII reduced leukocyte migration, the MPO activity and the amount of TNF-α and IL-1ß in the peritoneal fluid. In summary, these results indicate that RipII has an anti-inflammatory activity in chemical models of acute inflammation. RipII might be directly or indirectly inhibiting the activity, production or release of pro-inflammatory mediators involved in the generation of the pain associated with inflammation.