Antinociceptive Effect of Dillenia indica (Linn.) Mediated by Opioid and Cannabinoid Systems: Pharmacological and Chemical Studies.

Dillenia indica (Linn.) has been reported by several biological activities, including anti-inflammatory, antioxidant, antidiabetic, anti-hyperglycemic, antiproliferative, antimutagenic, anticholinesterase, and antimicrobial. In Brazilian traditional medicine, the fruits of D. indica have been used to treat general topical pain and inflammation, but with no scientific validation. Thus, aiming to study its chemical constitution and antinociceptive properties, the crude extract (CE) and fractions obtained from the fruits of D. indica were submitted to an in vivo pharmacological evaluation and a dereplication study by LC-MS/MS analysis, assisted by the Global Natural Product Social Molecular Networking (GNPS). The oral antinociceptive activity of the fruits of D. indica and the possible participation of the opioid and cannabinoid systems were demonstrated in the formalin-induced nociception model. The chemical dereplication study led us to identify several known chemical constituents, including flavonoids, such as caffeoylmalic acid, naringenin, quercetin, and kaempferol. According to literature data, our results are compatible with significant antinociceptive and anti-inflammatory activities. Therefore, the flavonoid constituents of the fruits of D. indica are probably responsible for its antioxidant, anti-inflammatory, and antinociceptive effects mediated by both opioid and cannabinoid systems, confirming its folk use in the treatment and relief of pain.

Lychnophora ericoides Mart. (Brazilian arnica) ethanol extract accelerates the skin wound healing process: Evidence for its mechanism of action.

BACKGROUND: Lychnophora ericoides Mart, also known as the Brazilian arnica or fake arnica, belongs to the Asteraceae family. Leaves and roots are used in alcoholic and hydroalcoholic preparations for the treatment of wounds, inflammation, and pain. PURPOSE: The present study aimed to investigate the effects of L. ericoides ethanolic extract (EELE) on cutaneous wound healing and the mechanisms of action involved. METHODS: A total of 72 C57BL/6 mice were randomly divided into four groups of six animals each. An excisional wound was made in the dorsal region of each mouse. The test groups were topically treated with the vehicle, a positive control commercial reference drug, EELE ointment (5%), and EELE ointment (10%). The treatments were applied over 14 days. The wound area was measured every two days to verify the wound closure kinetics. On days 3, 7, and 14 the wound tissue samples were processed for Hematoxylin and Eosin, Masson-Trichrome, and Toluidine blue staining. The expression of renin-angiotensin system (RAS) components, the vascular growth factor-A (VEGF-A), the basic fibroblast growth factor (FGF-2), and type I collagen genes were evaluated. Phytochemical analyses were performed using HPLC-DAD and HPLC-MS/MS. RESULTS: The EELE (10%) significantly reduced the wound area compared to the treatments used for the other groups. Histological analysis demonstrated that wounds treated with L. ericoides for 14 days developed improved anatomical skin features, healed with hair follicles and sebaceous glands, increased collagen production and angiogenesis, and decreased the number of mast cells at the injury site. Real-time PCR data demonstrated that groups treated with EELE (10%) showed increased Type I collagen, VEGF-A, FGF-2, and AT1R and decreased ACE II and receptor MAS. The healing action of L. ericoides may be related to the presence of phenolic compounds, such as phenolic acids, chlorogenic acid derivatives, and C-glycoside flavonoids. CONCLUSION: Topical treatment with EELE increases important factors for wound healing: FGF, VEGF, collagen formation, and the expression of the proliferative axis of the renin-angiotensin system. For the first time, the present study shows the healing action of L. ericoides at the molecular level in an animal model. This process can be used as an alternative therapy for wound healing and the development of herbal therapy.

Oxidation Products from the Neolignan Licarin A by Biomimetic Reactions and Assessment of in vivo Acute Toxicity.

Licarin A, a dihydrobenzofuranic neolignan presents in several medicinal plants and seeds of nutmeg, exhibits strong activity against protozoans responsible for Chagas disease and leishmaniasis. From biomimetic reactions by metalloporphyrin and Jacobsen catalysts, seven products were determined: four isomeric products yielded by epoxidation from licarin A, besides a new product yielded by a vicinal diol, a benzylic aldehyde, and an unsaturated aldehyde in the structure of the licarin A. The incubation with rat and human liver microsomes partially reproduced the biomimetic reactions by the production of the same epoxidized product of m/z 343 [M + H]+. In vivo acute toxicity assays of licarin A suggested liver toxicity based on biomarker enzymatic changes. However, microscopic analysis of tissues sections did not show any tissue damage as indicative of toxicity after 14 days of exposure. New metabolic pathways of the licarin A were identified after in vitro biomimetic oxidation reaction and in vitro metabolism by rat or human liver microsomes.

Neuroprotective Properties of Chlorogenic Acid and 4,5-Caffeoylquinic Acid from Brazilian arnica (Lychnophora ericoides) after Acute Retinal Ischemia.

Lychnophora is a genus of South American flowering plants in the daisy family, popularly known as "Brazilian arnica". It is used in traditional medicine as an anti-inflammatory and analgesic agent, whose active components are derived from chlorogenic acid (CGA) and C-flavonoids. Since the drugs currently used are ineffective to treat glaucoma, agents with antioxidant and anti-inflammatory properties may represent new alternatives in preventing cellular lesions in retinal ischemia. In this study, we report the neuroprotective effects of CGA and 4,5-di-O-[E]-caffeoylquinic (CQA) acid, isolated from Lychnophora plants, in a rodent glaucoma model. Wistar rats were administered intravitreally with 10 µg CGA or CGA, and then subjected to acute retinal ischemia (ISC) by increasing intraocular pressure (IPO) for 45 minutes followed (or not) by 15 minutes of reperfusion (I/R). Qualitative and quantitative analyses of neurodegeneration were performed using hematoxylin-eosin or Fluoro-Jade C staining protocols. All retinas submitted to ISC or I/R exhibited matrix disorganization, pyknotic nuclei, and pronounced vacuolization of the cytoplasm in the ganglion cell layer (GCL) and inner nuclear layer (INL). Pretreatment with CGA or CQA resulted in the protection of the retinal layers against matrix disorganization and a reduction in the number of vacuolized cells and pyknotic nuclei. Also, pretreatment with CGA or CQA resulted in a significant reduction in neuronal death in the GCL, the INL, and the outer nuclear layer (ONL) after ischemic insult. Our study demonstrated that CGA and CQA exhibit neuroprotective activities in retinas subjected to ISC and I/R induced by IPO in Wistar rats.

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.

Topical gel containing phenolic-rich extract from Ipomoea pes-capre leaf (Convolvulaceae) has anti-inflammatory, wound healing, and antiophidic properties.

The growing use of phytotherapy in clinical practice arouses interest in studies using medicinal plants as active ingredients for new medicines. Ipomoea pes-caprae has a wide medicinal use in the treatment of inflammatory disorders, skin wounds, stings, and painful rheumatic processes. Assayed in this study are the physicochemical characterization of a gel developed with this extract and the evaluation of its anti-inflammatory and healing efficacy, in addition to its antiedematogenic action on Bothrops snake envenoming in mice. The qualitative and quantitative analyses of the hydroethanolic extract by mass spectrometry showed 18 phenolic compounds, highlighting a high content of chlorogenic acid (0.92 µg/g), neochlorogenic acid (6.07 µg/g), and isochlorogenic acid (0.80 µg/g) compounds. The formulation was stable in relation to the physical-chemical characteristics during the time of analysis and was considered safe for topical treatment in animals, causing no skin irritation. Although the results have shown an absence of activity in the model of ear edema induced by croton oil (acute inflammation), the herbal gel efficiently inhibited carrageenan paw edema and chronic ear edema induced by multiple applications of croton oil, which may indicate the possible performance under the kinin pathway such as bradykinin, histamine, and serotonin. Wound healing in the group treated with the I. pes-caprae gel was accelerated compared with the placebo group, also confirmed through histological data. Edema induced by Bothrops erythromelas snake venom was efficiently reduced in the treatment with I. pes-caprae gel associated with the antibothropic-crotalic serum, whereas the antivenom alone was not effective. This approach presents a promising formulation based on I. pes-caprae with potential therapeutic use for inflammatory disorders.

Non-target molecular network and putative genes of flavonoid biosynthesis in Erythrina velutina Willd., a Brazilian semiarid native woody plant.

Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

Erythrina velutina Willd. alkaloids: Piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves.

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. Erythrina species are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids (BIAs), which can act on several pathology-related biological targets. Objectives: In this sense, in an unprecedented approach used with a non-model Fabaceae species grown in its unique arid natural habitat, a combined transcriptome and metabolome analyses (seeds and leaves) is presented. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a NextSeq 500 platform. Regarding metabolite profiling, the High-resolution Liquid Chromatography was coupled to DAD and a micrOTOF-QII mass spectrometer by using electrospray ionization (ESI) and Time of Flight (TOF) analyzer. The tandem MS/MS data were processed and analyzed through Molecular Networking approach. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids, several of them unique. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: Overall, these results could contribute by indicating potential biotechnological targets for modulation of erythrina alkaloids biosynthesis as well as improve molecular databases with omic data from a non-model medicinal plant, and reveal an interesting chemical diversity of Erythrina BIA harvested in Caatinga.

Identification of flavonoids in Hymenaea martiana Hayne (Fabaceae) by HPLC-DAD-MSn analysis.

Hymenaea martiana is a native tree known in Brazil as 'jatobá' and used in folk medicine to treat pain and inflammation. The aim of this work was to identify the flavonoids present in the crude ethanolic extract and ethyl acetate fraction using HPLC-DAD and LC-MSn analysis. The ethanolic extract was partitioned to obtain the ethyl acetate fraction. The analysis of astilbin content also was carried out by HPLC analysis. HPLC-DAD-ESI/MSn analysis of the ethanolic extract and ethyl acetate fraction revealed the presence of eleven peaks in the chromatograms, and all these peaks were identified: taxifolin, eucryphin, astilbin and 3 diastereoisomers, engeletin and 2 diastereoisomers, quercitrin and 2,6,3',4'-tetrahydroxy-2-benzylcoumaran-3-one. The ethyl acetate fraction had a higher astilbin concentration (151.87 µg/mL) than the ethanolic extract (40.13 µg/mL). In conclusion, the species could be considered a good source of flavonoids, which can be related to the main chemotaxonomic markers for the genus Hymenaea.

Anticancer Potential of Compounds from the Brazilian Blue Amazon.

"Blue Amazon" is used to designate the Brazilian Economic Exclusive Zone, which covers an area comparable in size to that of its green counterpart. Indeed, Brazil flaunts a coastline spanning 8000 km through tropical and temperate regions and hosting part of the organisms accredited for the country's megadiversity status. Still, biodiversity may be expressed at different scales of organization; besides species inventory, genetic characteristics of living beings and metabolic expression of their genes meet some of these other layers. These metabolites produced by terrestrial creatures traditionally and lately added to by those from marine organisms are recognized for their pharmaceutical value, since over 50% of small molecule-based medicines are related to natural products. Nonetheless, Brazil gives a modest contribution to the field of pharmacology and even less when considering marine pharmacology, which still lacks comprehensive in-depth assessments toward the bioactivity of marine compounds so far. Therefore, this review examined the last 40 years of Brazilian natural products research, focusing on molecules that evidenced anticancer potential-which represents ~ 15% of marine natural products isolated from Brazilian species. This review discusses the most promising compounds isolated from sponges, cnidarians, ascidians, and microbes in terms of their molecular targets and mechanisms of action. Wrapping up, the review delivers an outlook on the challenges that stand against developing groundbreaking natural products research in Brazil and on a means of surpassing these matters.

Time-Scale Shifting of Volatile Semiochemical Levels in Wild Type Lychnophora ericoides (Brazilian arnica) and Pollinator Records.

Lychnophora ericoides is a Brazilian folk phytomedicine from Cerrado's "campus rupestris". Its volatile organic compounds includes bisabolene-derivatives as major compounds. Herein we provide the chemical profiling of constitutive volatile sesquiterpenes from L. ericoides leaves, timeframe emissions surveys, and pollinators records. In situ samples of L. ericoides were harvested. A headspace-solid phase micro extraction method of pre-concentration was optimized. Identification was done through GC-MS. Isolation and structural elucidation were performed whenever necessary. Pollinators were registered in pictures and video. Short time-series and harmonic regressions determined rhythms of single compounds, and average chromatographic signal area was used to determine mono and sesquiterpene rhythms. Concluding, optimized headspace-solid phase micro extraction method of terpenes level analysis was reached. α-Pinene, ß-pinene, α-terpinene, para-cymene, limonene, γ-terpinene, terpinen-4-ol, dehydro-sesquicineole, and ß-guaiene were identified using GC-MS data. 11-dehydro cadinol and ortho-acetoxy bisabolol were elucidated. Sesquiterpenes concentrations were higher due to temperature rise, lower leaf age, and flowering seasons. Harmonic regressions determined that daylight might control levels of terpenes. Hummingbird, hemiptera insects, and wasps were recorded visiting Compositae capitulum for the first time. We studied nondomestic plants from in situ conditions and concluded that bisabolene-derivative levels were more abundant than monoterpenes during flowering throughout the summer.

Plant Natural Products for the Control of Aedes aegypti: The Main Vector of Important Arboviruses.

The mosquito species Aedes aegypti is one of the main vectors of arboviruses, including dengue, Zika and chikungunya. Considering the deficiency or absence of vaccines to prevent these diseases, vector control remains an important strategy. The use of plant natural product-based insecticides constitutes an alternative to chemical insecticides as they are degraded more easily and are less harmful to the environment, not to mention their lower toxicity to non-target insects. This review details plant species and their secondary metabolites that have demonstrated insecticidal properties (ovicidal, larvicidal, pupicidal, adulticidal, repellent and ovipositional effects) against the mosquito, together with their mechanisms of action. In particular, essential oils and some of their chemical constituents such as terpenoids and phenylpropanoids offer distinct advantages. Thiophenes, amides and alkaloids also possess high larvicidal and adulticidal activities, adding to the wealth of plant natural products with potential in vector control applications.

Sequesterpene Lactones Isolated from a Brazilian Cerrado Plant (Eremanthus spp.) as Anti-Proliferative Compounds, Characterized by Functional and Proteomic Analysis, are Candidates for New Therapeutics in Glioblastoma.

Gliomas are responsible for more than 60% of all primary brain tumors. Glioblastoma multiforme (GBM), a grade IV tumor (WHO), is one of the most frequent and malignant gliomas. Despite two decades of advances in the discovery of new markers for GBM, the chemotherapy of choice falls to temozolomide after surgery and radiotherapy, which are not enough to increase the survival of patients to more than 15 months. It is urgent to discover new anti-glioma compounds. Many compounds derived from natural products have been used in the development of anti-tumor drugs. In this work, we have screened six low molecular weight sesquiterpene lactones, isolated from Eremanthus spp., and studied their function as anti-proliferative agents against GBM strains. We demonstrated that two of them, goyazensolide and lychnofolide, were effective in reducing cell viability, preventing the formation of anchorage-dependent colony and were able to pass through a mimetic blood-brain barrier making them candidates for glioma therapy, being more potent than temozolomide, according to in vitro assays for the cell lines tested. Proteomic analysis revealed a number of altered proteins involved in glycolytic metabolism and cellular catabolism.

Fragmentation study of clerodane diterpenes from Casearia species by tandem mass spectrometry (quadrupole time-of-flight and ion trap).

RATIONALE: Clerodane-type diterpenes from Casearia species show important pharmacological activites such as antitumor, antimicrobial and anti-inflamatory. There are several mass spectrometry (MS)-based methods for identification of diterpenes; however, there is still a lack of MS procedures capable of providing characteristic fragmentation pathways for a rapid and unambiguous elucidation of casearin-like compounds. METHODS: Casearin-like compounds were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The fragmentation studies were carried out by tandem mass spectrometry in space (quadrupole time-of-flight (QTOF)) using different collision energies and also by tandem mass spectrometry in time (QIT) by selective isolation of product ions. RESULTS: Casearin-like compounds presented a predominance of sodium- and potassium-cationized precursor ions. Both QIT and QTOF techniques provided sequential neutral losses of esters related to the R1 to R5 substituents linked to the nucleus of the clerodane diterpenes. The fragmentation pathway is initiated with a cleavage of the ester moieties R2 followed by the elimination of the ester groups R3 , both losing neutral carboxylic acids. Using QIT, it was also possible to observe the cleavage of the ester groups R1 or R5 by MS4 experiments. CONCLUSIONS: Through a rational analysis of the fragmentation mechanisms of Casearia diterpenes it was possible to suggest an annotation strategy based on the sequential cleavages of the ester groups related to the R2 , R3 and R5 substituents. These results will assist studies of the dereplication and metabolomics involving casearin-like compounds present in complex extracts of Casearia species.

Yangambin and Epi-yangambin Isomers: New Purification Method from Ocotea fasciculata and First Cytotoxic Aspects Focusing on In Vivo Safety.

Ocotea fasciculata presents yangambin (YAN) and its isomer epi-yangambin (EPI-YAN) as major lignans, which are employed as the plant markers for quality control purposes and as potential pharmacological compounds. However, a gap between the pure isomers and safety and efficacy protocols is faced by the scientific community. In this context, this work aimed to report (i) a new and advantageous purifying process in a semi-preparative scale for YAN and EPI-YAN isolation from Ocotea fasciculata, and (ii) an in vitro cytotoxicity study to estimate, for the first time, the LD50 values of the isolated epimers, as well as the influence of albumin concentration in cell culture medium. The best condition for epimers isolation was achieved in normal-phase liquid chromatography. The lignan fraction (LF), previously obtained from the plant ethanolic extract, was purified yielding 17% and 29% of YAN and EPI-YAN, respectively. The in vitro study demonstrated that YAN and EPI-YAN were safe, and only at the highest concentration studied, a decrease on cell viability was observed. The estimated LD50 value was higher than 1612 mg/kg for both epimers. The LF, on the other hand, demonstrated an estimated LD50 of 422 mg/kg. Lignan cytotoxicity studies also evidenced that the higher cell viability was related to the higher concentration of fetal bovine serum as a source of albumin in medium. This is the first time the LD50 and safety of the isolated epimers were estimated, opening up great perspectives of success in in vivo studies.

Determination of phenolic compounds, in vitro antioxidant activity and characterization of secondary metabolites in different parts of Passiflora cincinnata by HPLC-DAD-MS/MS analysis.

Ethanol extracts of different parts of Passiflora cincinnata were obtained by maceration. The total phenolic and flavonoid contents were evaluated. The antioxidant activities were determined by ß-carotene-linoleic acid bleaching test, 2,2-diphenyl-1-picrylhydrazil (DPPH), and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging. The crude ethanol stem extract showed the highest amount of total polyphenols (45.53 mg gallic acid equivalent/g) while the highest total flavonoid contents (1.42 mg of quercetin equivalent/g) were observed in the leaf extract. The lowest IC50 (25.65 µg/ml) by the DPPH method was observed for the stem extract. The ABTS method showed a significant antioxidant activity for all investigated extracts. The secondary metabolite composition of ethanol extracts was assessed by HPLC-DAD-MS/MS analysis, leading to the identification of fourteen secondary metabolites in P. cincinnata extracts. These results showed the potentiality of this species as a source of phenolic compounds and antioxidants.

Characterization of aporphine alkaloids by electrospray ionization tandem mass spectrometry and density functional theory calculations.

RATIONALE: Aporphine alkaloids represent a large group of isoquinoline natural products with important roles in biological and biomedical areas. Their characterization by electrospray ionization tandem mass spectrometry (ESI-MS/MS) can contribute to their rapid identification in complex biological matrices. METHODS: We report the fragmentation of protonated 7,7-dimethylaporphine alkaloids by ESI-MS/MS, and the putative annotation of aporphine alkaloids in plant extracts. We used low- and high-resolution MS/MS analyses to rationalize the fragmentation pathways, and employed the B3LYP/6-31 + G(d,p) density functional theory (DFT) model to provide thermochemical parameters and to obtain the reactive sites. RESULTS: DFT calculations of a set of 7,7-dimethylaporphine alkaloids suggested the heterocyclic amino group as the most basic site due to the proton affinity of the nitrogen atom. Collision-induced dissociation experiments promoted • OCH3 elimination instead of the expected neutral loss of the heterocyclic amino group, pointing to the [M - 15 + H]•+ ion as the diagnostic fragment for 7,7-dimethylaporphine alkaloids. The analysis of plant extracts led to the annotation of 25 aporphine alkaloids. Their fragmentation initiated with the loss of the amino group followed by formation of a cyclic carbocation. Further reactions derived from consecutive charge-remote and/or charge-induced fragmentations of the substituents attached to the aromatic system. The mechanisms were re-examined based on plausible gas-phase ion chemistry reactions. CONCLUSIONS: Taken together, the diagnostic product ions and the series of radical and neutral eliminations provided information about the location of methylenedioxy, aromatic methoxy, and vicinal methoxy and hydroxy groups in aporphine alkaloids, assisting their characterization via MS/MS.

Characterization of 3-aminospirostane alkaloids from roots of Solanum paniculatum L. with hepatoprotective activity.

RATIONALE: Solanum paniculatum L., popularly known as jurubeba, has traditionally been used in Brazilian folk medicine for liver diseases. However, there is a lack of knowledge about the chemical characterization of 3-aminospirostane alkaloids, an important class related to pharmacological activities. This work aimed to characterize the alkaloids using liquid chromatography with tandem mass spectrometry (LC/MS/MS) supported by molecular networking and theoretical calculations as well as to evaluate the contribution to hepatoprotective activity. METHODS: S. paniculatum roots were collected and macerated with MeOH/H2 O (8:2) obtaining the crude extract (SP-CE). From this, partition using EtOAc with pH variation yielded the alkaloidic fraction (SP-AF). Both were evaluated in an acute liver injury model (100 and 200 mg/kg), after intraperitoneal administration of carbon tetrachloride (CCl4 ) in mice. AST (aspartate transaminase) and ALT (alanine transaminase) serum levels were investigated, as well as the histopathological characteristics. The SP-CE and SP-AF were analyzed by LC/MS/MS, using quadrupole/time-of-flight and ion-trap systems. The alkaloids annotated by the GNPS molecular network had their structures defined using gas-phase ionization and fragmentation reaction supported by theoretical calculations. RESULTS: The SP-CE and SP-AF decreased the ALT serum levels compared with the negative control. The group treated with the SP-CE (at the highest dose) demonstrated a significant decrease of ALT. Hepatic cell degeneration decrease was observed mainly at the highest dose of the treatment. Detailed electrospray ionization MS/MS data allowed us to identify alkaloids not previously reported, to propose their gas-phase reactions and to redefine the initial open ring fragmentation mechanism of the steroidal alkaloids with the jurubidine moiety. CONCLUSIONS: The results allowed us to identify seven steroidal alkaloids from jurubeba and redefine the initial mechanism of fragmentation. A significant hepatoprotective effect was also demonstrated, corroborating its traditional use.