Alkaloids from Siparuna (Siparunaceae) are predicted as the inhibitors of proteolysis and plasma coagulation caused by snake venom and potentially counteract phospholipase A2 activity of Bothrops jararaca.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. AIM OF THE STUDY: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. MATERIALS AND METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Myrtucommulones and Related Acylphloroglucinols from Myrtaceae as a Promising Source of Multitarget SARS-CoV-2 Cycle Inhibitors.

The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CLpro and PLpro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC50 was lower than 8.3 µg·mL-1). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H]-) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PLpro and 3CLpro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina, exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.

Antiviral, Cytoprotective, and Anti-Inflammatory Effect of Ampelozizyphus amazonicus Ducke Ethanolic Wood Extract on Chikungunya Virus Infection.

Chikungunya fever, a debilitating disease caused by Chikungunya virus (CHIKV), is characterized by a high fever of sudden onset and an intense arthralgia that impairs individual regular activities. Although most symptoms are self-limited, long-term persistent arthralgia is observed in 30-40% of infected individuals. Currently, there is no vaccine or specific treatment against CHIKV infection, so there is an urgent need for the discovery of new therapeutic options for CHIKF chronic cases. This present study aims to test the antiviral, cytoprotective, and anti-inflammatory activities of an ethanol extract (FF72) from Ampelozizyphus amazonicus Ducke wood, chemically characterized using mass spectrometry, which indicated the major presence of dammarane-type triterpenoid saponins. The major saponin in the extract, with a deprotonated molecule ion m/z 897 [M-H]-, was tentatively assigned as a jujubogenin triglycoside, a dammarane-type triterpenoid saponin. Treatment with FF72 resulted in a significant reduction in both virus replication and the production of infective virions in BHK-21-infected cells. The viability of infected cells was assessed using an MTT, and the result indicated that FF72 treatment was able to revert the toxicity mediated by CHIKV infection. In addition, FF72 had a direct effect on CHIKV, since the infectivity was completely abolished in the presence of the extract. FF72 treatment also reduced the expression of the major pro-inflammatory mediators overexpressed during CHIKV infection, such as IL-1ß, IL-6, IL-8, and MCP-1. Overall, the present study elucidates the potential of FF72 to become a promising candidate of herbal medicine for alphaviruses infections.

Anti-SARS-CoV-2 Activity of Ampelozizyphus amazonicus (Saracura-Mirá): Focus on the Modulation of the Spike-ACE2 Interaction by Chemically Characterized Bark Extracts by LC-DAD-APCI-MS/MS.

Traditional medicine shows several treatment protocols for COVID-19 based on natural products, revealing its potential as a possible source of anti-SARS-CoV-2 agents. Ampelozizyphus amazonicus is popularly used in the Brazilian Amazon as a fortifier and tonic, and recently, it has been reported to relieve COVID-19 symptoms. This work aimed to investigate the antiviral potential of A. amazonicus, focusing on the inhibition of spike and ACE2 receptor interaction, a key step in successful infection. Although saponins are the major compounds of this plant and often reported as its active principles, a polyphenol-rich extract was the best inhibitor of the spike and ACE2 interaction. Chemical characterization of A. amazonicus bark extracts by LC-DAD-APCI-MS/MS before and after clean-up steps for polyphenol removal showed that the latter play an essential role in maintaining this activity. The effects of the extracts on viral replication were also assessed, and all samples (aqueous and ethanol extracts) demonstrated in vitro activity, inhibiting viral titers in the supernatant of Calu-3 cells after 24 hpi. By acting both in the SARS-CoV-2 cell entry process and its replication, A. amazonicus bark extracts stand out as a multitarget agent, highlighting the species as a promising candidate in the development of anti-SARS-CoV-2 drugs.

In silico screening of phenylethanoid glycosides, a class of pharmacologically active compounds as natural inhibitors of SARS-CoV-2 proteases.

Since the advent of Covid-19, several natural products have been investigated regarding their in silico interactions with SARS-CoV-2 proteases - 3CLpro and PLpro, two of the most important pharmacological targets for antiviral development. Phenylethanoid glycosides (PG) are a class of natural products present in important medicinal plants and a drug containing this group of active ingredients has been successfully used in the treatment of Covid-19 in China. Thus, a dataset with 567 derivatives of this class was built from reviews published between 1994 and 2020, and their interaction against both SARS-CoV-2 proteases was investigated. The virtual screening was performed by filtering the PGs through the evaluation of scores based on the AutoDock Vina, GOLD/ChemPLP, and GOLD/GoldScore evaluation functions. The bRO5 pharmacokinetic parameters of the PGs ranked in the previous step were analyzed and their interaction with key amino acid residues of the 3CLpro and PLpro enzymes was evaluated. Ninety-eight compounds were identified by computational approaches against PLpro and 80 PGs against 3CLpro. Of these, four interacted with key catalytic residues of PLpro, which is an indicative of inhibitory activity, and three compounds interacted with catalytic key residues of 3CLpro. Of these, five PGs occur in plants of the Traditional Chinese Medicine (TCM), while two are components of plants/formulations currently used in the Covid-19 protocols in China. The data presented here show the potential of PGs as selective inhibitors of SARS-CoV-2 3CLpro and PLpro.

In vitro α-glucosidase inhibition by Brazilian medicinal plant extracts characterised by ultra-high performance liquid chromatography coupled to mass spectrometry.

Aiming at finding natural sources of antidiabetics agents, 15 extracts from Brazilian medicinal plants of the Atlantic Forest and Amazon region were tested against α-glucosidase enzyme. Plants were selected based on the taxonomic relationships with genera including several species with antidiabetic activity. In this screening, the extracts obtained from the flowers of Hyptis monticola and the leaves of Lantana trifolia and Lippia origanoides resulted endowed with promising anti-α-glucosidase activity. The extracts from H. monticola and from L. origanoides collected in two different areas, were characterised by ultra-high performance liquid chromatography coupled to mass spectrometry. Bioassay-guided fractionation led to the identification of several enzyme inhibiting compounds, among them the mechanism of action of naringenin and pinocembrin was investigated. The two L. origanoides extracts showed differences in bioactivity and in the phytochemical profiles. The fractionation of the extract from H. monticola led to a partial loss of the inhibitory effect.

Bioassay-Guided Fractionation of Siparuna glycycarpa n-Butanol Extract with Inhibitory Activity against Influenza A(H1N1)pdm09 Virus by Centrifugal Partition Chromatography (CPC).

Siparuna glycycarpa occurs in the Amazon region, and some species of this genus are used in Brazilian folk medicine. A recent study showed the inhibitory effect of this species against influenza A(H1N1)pdm09 virus, and in order to acquire active fractions, a polar solvent system n-butanol-methanol-water (9:1:10, v/v) was selected and used for bioassay-guided fractionation of n-butanol extract by centrifugal partition chromatography (CPC). The upper phase was used as stationary phase and the lower phase as mobile (descending mode). Among the collected fractions, the ones coded SGA, SGC, SGD, and SGO showed the highest antiviral inhibition levels (above 74%) at 100 µg·mL-1 after 24 h of infection. The bioactive fractions chemical profiles were investigated by LC-HRMS/MS data in positive and negative ionization modes exploring the Global Natural Products Social Molecular Networking (GNPS) platform to build a molecular network. Benzylisoquinoline alkaloids were annotated in the fractions coded SGA, SGC, and SGD collected during elution step. Aporphine alkaloids, O-glycosylated flavonoids, and dihydrochalcones in SGO were acquired with the change of mobile phase from lower aqueous to upper organic. Benzylisoquinolinic and aporphine alkaloids as well as glycosylated flavonoids were annotated in the most bioactive fractions suggesting this group of compounds as responsible for antiviral activity.

Saracura-Mirá, a Proposed Brazilian Amazonian Adaptogen from Ampelozizyphus amazonicus.

The Amazon Forest is known all over the world for its diversity and exuberance, and for sheltering several indigenous groups and other traditional communities. There, as well as in several other countries, in traditional medical systems, weakness, fatigue and debility are seen as limiting health conditions where medicinal plants are often used in a non-specific way to improve body functions. This review brings together literature data on Ampelozizyphus amazonicus, commonly known in Brazil as "saracura-mirá" and/or "cerveja de índio", as an Amazonian adaptogen, including some contributions from the authors based on their ethnographic and laboratory experiences. Topics such as botany, chemistry, ethnopharmacological and pharmacological aspects that support the adaptogen character of this plant, as well as cultivation, market status and supply chain aspects are discussed, and the gaps to establish "saracura-mirá" as an ingredient for the pharmaceutical purposes identified. The revised data presented good scientific evidence supporting the use of this Amazonian plant as a new adaptogen. Literature data also reveal that a detailed survey on natural populations of this plant is needed, as well as agronomical studies that could furnish A. amazonicus bark as a raw material. Another important issue is the lack of developed quality control methods to assure its quality assessment.

Flavonoids from Siparuna cristata as Potential Inhibitors of SARS-CoV-2 Replication.

The novel coronavirus SARS-CoV-2 has been affecting the world, causing severe pneumonia and acute respiratory syndrome, leading people to death. Therefore, the search for anti-SARS-CoV-2 compounds is pivotal for public health. Natural products may present sources of bioactive compounds; among them, flavonoids are known in literature for their antiviral activity. Siparuna species are used in Brazilian folk medicine for the treatment of colds and flu. This work describes the isolation of 3,3',4'-tri-O-methyl-quercetin, 3,7,3',4'-tetra-O-methyl-quercetin (retusin), and 3,7-di-O-methyl-kaempferol (kumatakenin) from the dichloromethane extract of leaves of Siparuna cristata (Poepp. & Endl.) A.DC., Siparunaceae, using high-speed countercurrent chromatography in addition to the investigation of their inhibitory effect against SARS-CoV-2 viral replication. Retusin and kumatakenin inhibited SARS-CoV-2 replication in Vero E6 and Calu-3 cells, with a selective index greater than lopinavir/ritonavir and chloroquine, used as control. Flavonoids and their derivatives may stand for target compounds to be tested in future clinical trials to enrich the drug arsenal against coronavirus infections. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43450-021-00162-5.

Distribution of 5,6-dihydro-α-pyrones by electrospray ionization ion trap mass spectrometry in different aerial parts of Hyptis monticola.

Hyptis monticola Mart. ex Benth. (Lamiaceae) is an endemic species of altitude regions of Brazil. From the leaves of this plant, two 5,6-dihydro-α-pyrones, named monticolides A and B, have been reported as cytotoxic agents against different tumor cell lines. The isolation by high-speed countercurrent chromatography in combination with recycling preparative high-performance liquid chromatography of the undescribed monticolides C-F is presented. These compounds corresponded to a series of related monticolide derivatives differing from each other by the number of acyl substituents. Their characterization by mass spectrometry and nuclear magnetic resonance is also presented, in conjunction with an evidence by a simple chemical correlation for their absolute stereochemistry. The distribution of these chemical markers in extracts of flowers, leaves and branches collected in different seasons by electrospray ionization ion trap mass spectrometry in positive mode was analyzed. Multivariate data analyses indicated that seasonality affects monticolide concentrations in different organs of the aerial parts. Monticolides A-F seem to be present as the original markers of the analyzed plant. However, mono-, di- and triacetylated monticolides can undergo acid-catalyzed transesterifications and their natural yields estimated were affected during the isolation procedures.

Amazonian Siparuna extracts as potential anti-influenza agents: Metabolic fingerprinting.

ETHNOPHARMACOLOGICAL RELEVANCE: Siparuna species are used in Brazilian Folk Medicine for the treatment and prophylaxis of colds, fever, headache, gastrointestinal disorders and rheumatic pain. AIM OF THE STUDY: This study aimed to investigate a possible anti-influenza activity of 25 extracts from leaves of Amazonian S. cristata, S. decipiens, S. glycycarpa, S. reginae and S. sarmentosa based on their folk medicinal uses as well as to investigate their metabolic fingerprinting. The chemical composition of the active extracts was further dereplicated. MATERIAL AND METHODS: The chemical composition of the crude EtOH extracts from five Siparuna species were investigated by ESI (±) LC-QTOF-MS2. Organic extracts were obtained by liquid-liquid partition with solvents of increasing polarity, generating 25 extracts which were subjected to a quick DI-ESI (±) IT-MS fingerprint analysis. These extracts were tested against influenza virus replication and cellular toxicity using MDCK cells and influenza A/Michigan/45/2015 (H1N1)pdm09 virus. The compounds in the active BuOH extracts from S. glycycarpa and S. sarmentosa were annotated by ESI (±) LC-QTOF-MS2. RESULTS: Analysis of the EtOH extracts revealed the presence of alkaloids and flavonoids, in the positive and negative ionization modes. Out of the 25 organic extracts screened for their antiviral activity, the BuOH extracts from S. glycycarpa and S. sarmentosa were the most active, inhibiting 96.0 ± 1.3% and 89.5 ± 0.8% of influenza virus replication 24 h post-infection. These inhibitory effects were maintained until 72hpi. Alkaloids, O- and C-flavonoid glycosides, dihydrochalcones and a procyanidin dimer were annotated in these extracts. CONCLUSIONS: The inhibitory effect against influenza A(H1N1)pdm09 virus replication shown by Amazonian Siparuna species corroborates the use of these plants in Brazilian Folk Medicine, showing their potential as anti-influenza agents. These promising results stimulate the continuation of this study with the aim of isolating the compound(s) responsible for this bioactivity, thus contributing to a better knowledge of those species and to the research of natural products with potential anti-influenza activity.

Antifungal Phenylpropanoid Glycosides from Lippia rubella.

Lippia species share various pharmacological activities and are used in traditional cooking and medicine worldwide. Combined chromatographic techniques such as column chromatography, high-performance liquid chromatography, and countercurrent chromatography led to the purification of two new antifungal phenylpropanoid glycosides, lippiarubelloside A (1) and lippiarubelloside B (2), by bioactivity-directed fractionation of an ethanol-soluble extract from Lippia rubella, in addition to the known active related compounds forsythoside A (3), verbascoside (4), isoverbascoside (5), and poliumoside (6). The structures of compounds 1 and 2 were determined by comparison of their NMR spectroscopic data with the prototype active compound 4. Cryptococcus neoformans, which causes opportunistic lung infections, was sensitive to compounds 1-6 in the concentration range of 15-125 µg/mL. A synergistic effect (FICindex = 0.5) between 3 and amphotericin B was demonstrated. The glycosylated flavonoids pectolinarin (7), linarin (8), and siparunoside (9) were also isolated.

Preparative mass-spectrometry profiling of minor concentrated metabolites in Salicornia gaudichaudiana Moq by high-speed countercurrent chromatography and off-line electrospray mass-spectrometry injection.

Salicornia species have just been introduced to the European market as a vegetable named 'samphire', 'green asparagus', or 'sea asparagus'. Due to its increasing attention, and associated value, minor compounds of Salicornia gaudichaudiana Moq were investigated. The use of countercurrent chromatography and mass spectrometry enabled the search for known, as well as potentially novel natural products. Their identification was achieved based on molecular weights and mass-spectrometric fragmentation data. Low detection limits enabled the visualization of all compounds with their identification in almost real time close to the preparative countercurrent chromatography experiment. A list of known natural products from Salicornia genus guided the identification process of compounds occurring in Salicornia gaudichaudiana Moq by tandem mass spectrometry fragment comparison. The natural product classes were divided into four groups: chlorogenic acid derivatives; flavonoid derivatives; pentacyclic triterpenoid saponins; and other compounds.

Structure Elucidation, Conformation, and Configuration of Cytotoxic 6-Heptyl-5,6-dihydro-2 H-pyran-2-ones from Hyptis Species and Their Molecular Docking to α-Tubulin.

Cytotoxic 6-heptyl-5,6-dihydro-2 H-pyran-2-ones are chemical markers of Hyptis (Lamiaceae) and are responsible for some of the therapeutic properties of species with relevance to traditional medicine. The present investigation describes the isolation of known pectinolides A-C (1-3), in addition to the new pectinolides I-M (4-8), from two Mexican collections of H. pectinata by HPLC. The novel biosynthetically related monticolides A (9) and B (10) were also isolated by high-speed countercurrent chromatography from H. monticola, an endemic species of the Brazilian southeastern high-altitude regions. A combination of chemical correlations, chiroptical measurements, and Mosher ester NMR analysis was used to confirm their absolute configuration. The utility of DFT-NMR chemical shifts and JH-H calculations was assessed for epimer differentiation. Molecular docking studies indicated that 6-heptyl-5,6-dihydro-2 H-pyran-2-ones have a high affinity for the pironetin-binding site of α-tubulin, which may be a possible mechanism contributing to the cytotoxic potential of these small and flexible molecules.

Use of counter-current chromatography as a selective extractor for the diterpenequinone 7α-hydroxyroyleanone from Tetradenia riparia.

A solvent system was developed for selective isolation by high-speed counter-current chromatography (HSCCC) of the benzoquinone 7α-hydroxyroileanone, 1, a bioactive diterpene from a dichloromethane extract of Tetradenia riparia leaves. Several solvent systems were initially studied, including hexane-ethyl acetate-methanol-water in several ratios, hexane-acetone-methanol-water, hexane-ethanol-water and hexane-acetonitrile-methanol, which gave recovery rates for the target compound between 13.4 and 35.9%. The new solvent system hexane-5% aqueous Na2CO3 (1:1) was developed based on the chemical ionization reaction of the benzoquinone hydroxyl group in the basic pH of the carbonate solution, prompted by the extraction procedure used for the extraction of lapachol (a natural naphtoquinone) from a Tabebuia species wood. By using the HSCCC chromatograph as a liquid-liquid extractor with the above mentioned solvent system the recovery rate of 1 increased to 81.8%, affording the quinone with 97% purity.

Counter-current chromatography with off-line detection by ultra high performance liquid chromatography/high resolution mass spectrometry in the study of the phenolic profile of Lippia origanoides.

Lippia origanoides (Verbenaceae) is an important Brazilian medicinal plant, also used for culinary purposes. Most chemical studies with this plant have been focused on its volatile composition. In this work, we combined High-Speed Counter-current Chromatography (HSCCC) and High Performance Liquid Chromatography coupled to Ultra Violet detection and High Resolution Mass Spectrometry (HPLC-UV-HRMSn) methodologies to access the non-volatile chemical composition of L. origanoides. The crude ethanol extract of L. origanoides (LOEF) was first analyzed by HPLC-UV-HRMSn and allowed the identification of 7 major compounds. Among them, eriodictyol, naringenin and pinocembrin, were determined and are phytochemical markers of this plant. However, owing to the complexity of this plant matrix, LOEF was fractionated by HSCCC (hexane-ethanol-water, 4:3:1) as a tool for preparative pre-purification, affording a flavonoid-rich fraction. A column screening with the chromatographic stationary phases ZIC-HILIC, monolithic and particulate RP18 was performed. The best column separation was achieved with a Purospher STAR RP18e, which was used for HPLC-DAD-HRMSn studies. By this approach 12 compounds were further identified in addition to the major ones identified in the raw extract. Two of them, 6,8-di-C-hexosyl-luteolin and 6,8-di-C-glucosyl-apigenin, are being reported for the first time in the family Verbenaceae. This work shows the integration of HSCCC as a preparative tool for the fractionation and purification of natural products from a complex plant extract with other analytical techniques, with the purpose of showing each technique's potential.

Schinus terebinthifolius countercurrent chromatography (Part II): Intra-apparatus scale-up and inter-apparatus method transfer.

Countercurrent chromatography (CCC) is being widely used across the world for purification of various materials, especially in natural product research. The predictability of CCC scale-up has been successfully demonstrated using specially designed instruments of the same manufacturer. The reality is that the most of CCC users do not have access to such instruments and do not have enough experience to transfer methods from one CCC column to another. This unique study of three international teams is based on innovative approach to simplify the scale-up between different CCC machines using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. The optimized separation methodology, recently developed by the authors (Part I), was repeatedly performed on CCC columns of different design available at most research laboratories across the world. Hexane - ethyl acetate - methanol - water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3ß-masticadienolic acids as target compounds to monitor stationary phase retention and calculate peak resolution. It has been demonstrated that volumetric, linear and length scale-up transfer factors based on column characteristics can be directly applied to different i.d., volume and length columns independently on instrument make in an intra-apparatus scale-up and inter-apparatus method transfer.

A new tropane alkaloid from the leaves of Erythroxylum subsessile isolated by pH-zone-refining counter-current chromatography.

Tropane alkaloids are bioactive metabolites with great importance in the pharmaceutical industry and the most important class of natural products found in the Erythroxylum genus. However, these compounds are usually separated by traditional chromatographic techniques, in which the sample is progressively purified in multiple chromatographic steps, resulting in a time- and solvent-consuming procedure. In this work we present the isolation of a novel alkaloid, 6ß,7ß-dibenzoyloxytropan-3α-ol, together with the two known 3α-benzoyloxynortropan-6ß-ol and 3α,6ß-dibenzoyloxytropane alkaloids, directly from the crude alkaloid fraction from the leaves of Erythroxylum subsessile, by using a single run pH-zone-refining counter-current chromatography method. The ethyl acetate/water (1:1, v/v) biphasic solvent system with triethylamine and HCl as retention and eluter agents, respectively, was used to isolate tropane alkaloids for the first time. The structures of the isolated alkaloids were elucidated by spectroscopic methods.

Spray-dried extract from the Amazonian adaptogenic plant Ampelozizyphus amazonicus Ducke (Saracura-mirá): Chemical composition and immunomodulatory properties.

Ampelozizyphus amazonicus Ducke is a medicinal plant used in the Amazon region to prepare a drink with tonic, immunomodulatory and adaptogenic properties. Due to the growing interest in dietary supplements with these properties and, to provide a new functional ingredient, barks from A. amazonicus were extracted. The extract was spray dried without drying adjuvants, resulting in a powder (SARF), which was characterized by its physico-chemical properties and proximate, mineral and saponin contents. The SARF saponins were characterized by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry (HPLC-HRMSn) analysis. The SARF particles tended to have a spherical shape and a unimodal size distribution. The particles also had good rehydration characteristics and high saponin content (33%). The effect of SARF on antibody production was investigated, and we found that SARF increased the basal levels of anti-ovalbumin, anti-LPS and anti-dextran IgM antibodies, and the anti-dextran IgG antibodies in unimmunized mice. No increase in antibody titers was observed after SARF treatment in immunized mice. These results suggest that SARF could be an interesting new functional ingredient for food applications or pharmaceutical products.

Gradient Elution in Countercurrent Chromatography.

Countercurrent chromatography is a form of liquid-liquid partition chromatography in which the stationary liquid phase is retained in the apparatus without the use of a solid support. Gradient elution in countercurrent chromatography can be used in many different ways, such as linear gradients, stepwise elution gradients, pH gradients, etc. The main goal of using the gradient approach is to shorten the duration of the separation and improve resolution, especially when the retention range of the sample to be purified is broadening and, thus, the compounds cannot be purified by only one solvent system. The principle is based on modifying the mobile phase to increase the elution strength with no or minimum changes in the stationary phase, which can be a difficult task since both phases are in intimate contact all the time. The most common ways to perform gradients in countercurrent chromatography are changing the mobile's phase polarity, flow rate, and pH, what is called linear or step gradient, flow rate gradient, pH gradient, respectively.