Bioprospecting-based untargeted metabolomics identifies alkaloids as potential anti-inflammatory bioactive markers of Ocotea species (Lauraceae).

BACKGROUND: Species within the Ocotea genus (Lauraceae), have demonstrated an interesting profile of bioactivities. Renowned for their diverse morphology and intricate specialized metabolite composition, Ocotea species have re-emerged as compelling candidates for bioprospecting in drug discovery research. However, it is a genus insufficiently studied, particularly regarding anti-inflammatory activity. PURPOSE: To investigate the anti-inflammatory activity of Ocotea spp. extracts and determine the major markers in this genus. METHODS: Extracts of 60 different Ocotea spp. were analysed by an ex vivo anti-inflammatory assay in human whole blood. The experiment estimates the prostaglandin E2 levels, which is one of the main mediators of the inflammatory cascade, responsible for the classical symptoms of fever, pain, and other common effects of the inflammatory process. Untargeted metabolomics analysis through liquid chromatography coupled with high-resolution mass spectrometry was performed, along with statistical analysis, to investigate which Ocotea metabolites are correlated with their anti-inflammatory activity. RESULTS: The anti-inflammatory screening indicated that 49 out of 60 Ocotea spp. extracts exhibited significant inhibition of PGE2 release compared to the vehicle (p < 0.05). Furthermore, 10 of these extracts showed statistical similarity to the reference drugs. The bioactive markers were accurately identified using multivariate statistics combined with a fold change (> 1.5) and adjusted false discovery rate analysis as unknown compounds and alkaloids, with a majority of aporphine and benzylisoquinolines. These alkaloids were annotated with an increased level of confidence since MSE spectra were compared with comprehensive databases. CONCLUSION: This study represents the first bioprospecting report revealing the anti-inflammatory potential of several Ocotea spp. The determination of their anti-inflammatory markers could contribute to drug discovery and the chemical knowledge of the Ocotea genus.

Profile of Myracrodruon urundeuva Volatile Compounds Ease of Extraction and Biodegradability and In Silico Evaluation of Their Interactions with COX-1 and iNOS.

Myracrodruon urundeuva Fr. Allem. (Anacardiaceae) is a tree popularly known as the "aroeira-do-sertão", native to the caatinga and cerrado biomes, with a natural dispersion ranging from the Northeast, Midwest, to Southeast Brazil. Its wood is highly valued and overexploited, due to its characteristics such as durability and resistance to decaying. The diversity of chemical constituents in aroeira seed has shown biological properties against microorganisms and helminths. As such, this work aimed to identify the profile of volatile compounds present in aroeira seeds. Headspace solid phase microextraction was employed (HS-SPME) using semi-polar polydimethylsiloxane-divinylbenzene fiber (PDMS/DVB) for the extraction of VOCs. 22 volatile organic compounds were identified: nine monoterpenes and eight sesquiterpenes, in addition to six compounds belonging to different chemical classes such as fatty acids, terpenoids, salicylates and others. Those that stood out were p-mentha-1,4, 4(8)-diene, 3-carene (found in all samples), caryophyllene and cis-geranylacetone. A virtual docking analysis suggested that around 65% of the VOCs molar content from the aroeiras seeds present moderate a strong ability to bind to cyclooxygenase I (COX-I) active site, oxide nitric synthase (iNOS) active site (iNOSas) or to iNOS cofactor site (iNOScs), corroborating an anti-inflamatory potential. A pharmacophoric descriptor analysis allowed to infer the more determinant characteristics of these compounds' conferring affinity to each site. Taken together, our results illustrate the high applicability for the integrated use of SPME, in silico virtual screening and chemoinformatics tools at the profiling of the biotechnological and pharmaceutical potential of natural sources.

Optimization of Extraction Conditions and Characterization of Volatile Organic Compounds of Eugenia klotzschiana O. Berg Fruit Pulp.

Eugenia klotzschiana O. Berg is a native species to the Cerrado biome with significant nutritional value. However, its volatile organic compounds (VOCs) chemical profile is not reported in the scientific literature. VOCs are low molecular weight chemical compounds capable of conferring aroma to fruit, constituting quality markers, and participating in the maintenance and preservation of fruit species. This work studied and determined the best conditions for extraction and analysis of VOCs from the pulp of Eugenia klotzschiana O. Berg fruit and identified and characterized its aroma. Headspace solid-phase microextraction (HS-SPME) was employed using different fiber sorbents: DVB/CAR/PDMS, PDMS/DVB, and PA. Gas chromatography and mass spectrometry (GC-MS) were employed to separate, detect, and identify VOCs. Variables of time and temperature of extraction and sample weight distinctly influenced the extraction of volatiles for each fiber. PDMS/DVB was the most efficient, followed by PA and CAR/PDMS/DVB. Thirty-eight compounds that comprise the aroma were identified among sesquiterpenes (56.4%) and monoterpenes (30.8%), such as α-fenchene, guaiol, globulol, α-muurolene, γ-himachalene, α-pinene, γ-elemene, and patchoulene.

Paper Spray Mass Spectrometry on the Analysis of Phenolic Compounds in Rhynchelytrum repens: A Tropical Grass with Hypoglycemic Activity.

The characterization of plant compounds with pharmacological activity is a field of great relevance in research and development. As such, identification techniques with the goal of developing new drugs or even validating the bioactive properties of extracts must be explored in order to further expand the knowledge of plant extract composition. Most works in this field employ HPLC, when exploring non-structural and cell wall carbohydrates from Rhynchelytrum repens. Phenolic compounds were studied by classical chromatography techniques and UV-vis spectrophotometry, with C-glycosylated flavonoids being detected but with no further details regarding the chemical structure of these compounds. In this work we employ paper spray ionization mass spectrometry (PS-MS) for the evaluation of the chemical profile of R. repens methanol extract. Positive ionization mode identified 15 compounds, belonging to flavonoids, fatty acids, and other classes of compounds; negative mode ionization was able to identify 20 compounds comprising the classes of quinic acids, stilbenes and flavonoids. PS-MS proved effective for the evaluation of R. repens extracts, making it possible to identify a total of thirty-five compounds. The bioactive properties attributed to R. repens were confirmed by the identification and characterization of compounds identified by PS-MS.

Neuroprotective potential of Ayahuasca and untargeted metabolomics analyses: applicability to Parkinson's disease.

ETHNOPHARMACOLOGY RELEVANCE: Ayahuasca is a tea produced through decoction of Amazonian plants. It has been used for centuries by indigenous people of South America. The beverage is considered to be an ethnomedicine, and it is traditionally used for the treatment of a wide range of diseases, including neurological illness. Besides, some scientific evidence suggests it may be applicable to Parkinson's disease (PD) treatment. Thus, Ayahuasca deserves in depth studies to clarify its potential role in this disease. AIM OF THE STUDY: This study aimed to use an untargeted metabolomics approach to evaluate the neuroprotective potential of the Ayahuasca beverage, the extracts from its matrix plants (Banisteriopsis caapi and Psychotria viridis), its fractions and its main alkaloids on the viability of SH-SY5Y neuroblastoma cells in an in vitro PD model. MATERIAL AND METHODS: The cytotoxicity of Ayahuasca, crude extracts, and fractions of B. caapi and P. viridis, as well as neuroprotection promoted by these samples in a 6-hydroxydopamine (6-OHDA)-induced neurodegeneration model, were evaluated by the MTT assay at two time-points: 48 h (T1) and 72 h (T2). The main alkaloids from Ayahuasca matrix plants, harmine (HRE) and N,N-dimethyltryptamine (DMT), were also isolated and evaluated. An untargeted metabolomics approach was developed to explore the chemical composition of samples with neuroprotective activity. Ultra-Performance Liquid Chromatography coupled to Electrospray Ionisation and Time-of-Flight (UPLC-ESI-TOF) metabolome data was treated and further analysed using multivariate statistical analyses (MSA): principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The metabolites were dereplicated using the Dictionary of Natural Products and an in house database. The main alkaloids were also quantified by UPLC-MS/MS. RESULTS: The samples did not cause cytotoxicity in vitro and three of samples intensely increased cell viability at T1. The crude extracts, alkaloid fractions and HRE demonstrated remarkable neuroprotective effect at T2 while the hydroalcoholic fractions demonstrated this neuroprotective effect at T1 and T2. Several compounds from different classes, such as ß-carbolines and monoterpene indole alkaloids (MIAs) were revealed correlated with this property by MSA. Additionally, a total of 2419 compounds were detected in both ionisation modes. HRE showed potent neuroprotective action at 72 h, but it was not among the metabolites positively correlated with the most efficacious neuroprotective profile at either time (T1 and T2). Furthermore, DMT was statistically important to differentiate the dataset (VIP value > 1), although it did not exhibit sufficient neuroprotective activity by in vitro assay, neither a positive correlation with T1 and T2 neuroprotective profile, which corroborated the MSA results. CONCLUSION: The lower doses of the active samples stimulated neuronal cell proliferation and/or displayed the most efficacious neuroprotection profile, namely by preventing neuronal damage and improving cell viability against 6-OHDA-induced toxicity. Intriguingly, the hydroalcoholic fractions exhibited enhanced neuroprotective effects when compared to other samples and isolated alkaloids. This finding corroborates the significance of a holistic approach. The results demonstrate that Ayahuasca and its base plants have potential applicability for PD treatment and to prevent its progression differently from current drugs to treat PD.

Anti-urolithiatic and anti-inflammatory activities through a different mechanism of actions of Cissus gongylodes corroborated its ethnopharmacological historic.

ETHNOPHARMACOLOGICAL RELEVANCE: Species Cissus gongylodes has been used in the traditional medicine in South America and India for the treatment of urolithiasis, biliary and inflammatory problems without any scientific evidence. AIM OF THE STUDY: This work was developed to investigate for the first time the anti-inflammatory and anti-urolithiatic activities of leaf decoction of C. gongylodes. MATERIALS AND METHODS: Decoction was subjected to anti-inflammatory evaluation by the in vivo assay of ear oedema and quantification of the main mediators of inflammation PGE2 and LTB4, and the cytokine TNF-α. The decoction's anti-urolithiatic activity was determined by different in vitro assays to evaluate the inhibition and dissolution of the most prevalent types of kidney stones: calcium oxalate (CaOx) and struvite. Diffusion in gel technique and fresh urine of a patient with renal stone were used to investigate the inhibition and dissolution of CaOx, respectively, and the single diffusion gel growth technique was used to evaluate the inhibition and dissolution of struvite crystals. The decoction was chemically characterized by UHPLC-ESI-HRMS analysis. RESULTS: Decoction showed in vivo anti-inflammatory activity by potent decreasing the level of both the main mediators of inflammation and dose-dependent in vitro anti-urolithiatic action by inhibition and dissolution of both type of crystals, CaOx and struvite. CONCLUSIONS: Results obtained corroborate the reports of the traditional use of the decoction of Cissus gongylodes. Besides, it showed multi-target mechanisms actions, inhibition of the main inflammatory pathways, and inhibition/dissolution of the most prevalent types of crystals on urolithiasis. These actions make the decoction a promissory source to the development of new and more efficient drugs.

Improved vascularisation but inefficient in vivo bone regeneration of adipose stem cells and poly-3-hydroxybutyrate-co-3-hydroxyvalerate scaffolds in xeno-free conditions.

Bone defects are a common clinical situation. However, bone regeneration remains a challenge and faces the limitation of poor engraftment due to deficient vascularisation. Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and human adipose stem cells (hASC) are promising for vascularisation and bone regeneration. Therefore, we sought to investigate the bone regenerative capacity of hASCs cultured in allogeneic human serum (aHS) and PHB-HV scaffolds in a nude mouse model of the critical-sized calvarial defect. We evaluated bone healing for three treatment groups: empty (control), PHB-HV and PHB-HV + hASCs. The pre-implant analysis showed that hASCs colonised the PHB-HV scaffolds maintaining cell viability before implantation. Histological analysis revealed that PHB-HV scaffolds were tolerated in vivo; they integrated with adjacent tissue eliciting a response like a foreign body reaction, and tiny primary bone was observed only in the PHB-HV group. Also, the µ-CT analysis revealed only approximately 10% of new bone in the bone defect area in both the PHB-HV and PHB-HV + hASCs groups. The expression of BGLAP and its protein (osteocalcin) by PHB-HV + hASCs group and native bone was similar while the other bone markers RUNX2, ALPL and COL1A1 were upregulated, but this expression remained significantly lower compared to the native bone. Nevertheless, the PHB-HV group showed neovascularisation at 12 weeks post-implantation while PHB-HV + hASCs group also exhibited higher VEGFA expression as well as a higher number of vessels at 4 weeks post-implantation, and, consequently, earlier neovascularisation. This neovascularisation must be due to scaffold architecture, improved by hASCs, that survived for the long term in vivo in the PHB-HV + hASCs group. These results demonstrated that hASCs cultured in aHS combined with PHB-HV scaffolds were ineffective to promote bone regeneration, although the construct of hASCs + PHB-HV in xeno-free conditions improved scaffold vascularisation representing a strategy potentially promising for other tissue engineering applications.

Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation.

INTRODUCTION: Human adipose tissue-derived stem cells (hASCs) are attractive cells for therapeutic applications and are currently being evaluated in multiple clinical trials. Prior to their clinical application, hASCs must be expanded ex vivo to obtain the required number of cells for transplantation. Fetal bovine serum is the supplement most widely used for cell culture, but it has disadvantages and it is not safe for cell therapy due to the risks of pathogen transmission and immune reaction. Furthermore, the cell expansion poses a risk of accumulating genetic abnormalities that could lead to malignant cell transformation. In this study, our aim was to evaluate the proliferation pattern as well as the resistance to spontaneous transformation of hASCs during expansion in a xeno-free culture condition. METHODS: hASCs were expanded in Dulbecco's modified Eagle's medium supplemented with pooled allogeneic human serum or fetal bovine serum to enable a side-by-side comparison. Cell viability and differentiation capacity toward the mesenchymal lineages were assessed, along with immunophenotype. Ki-67 expression and the proliferation kinetics were investigated. The expression of the transcription factors c-FOS and c-MYC was examined with Western blot, and MYC, CDKN2A, ERBB2 and TERT gene expression was assessed with quantitative PCR. Senescence was evaluated by ß-gal staining. Karyotype analysis was performed and tumorigenesis assay in vivo was also evaluated. RESULTS: The hASCs expanded in medium with pooled allogeneic human serum did not show remarkable differences in morphology, viability, differentiation capacity or immunophenotype. The main difference observed was a significantly higher proliferative effect on hASCs cultured in pooled allogeneic human serum. There was no significant difference in C-FOS expression; however, C-MYC protein expression was enhanced in pooled allogeneic human serum cultures compared to fetal bovine serum cultures. No difference was observed in MYC and TERT mRNA levels. Moreover, the hASCs presented normal karyotype undergoing senescence, and did not form in vivo tumors, eliminating the possibility that spontaneous immortalization of hASCs had occurred with pooled allogeneic human serum. CONCLUSIONS: This complete characterization of hASCs cultivated in pooled allogeneic human serum, a suitable xeno-free approach, shows that pooled allogeneic human serum provides a high proliferation rate, which can be attributed for the first time to C-MYC protein expression, and showed cell stability for safe clinical applications in compliance with good manufacturing practice.