Antiviral Effect of Natural and Semisynthetic Diterpenoids against Adenovirus Infection in vitro.

The emergence and re-emergence of viruses has highlighted the need to develop new broad-spectrum antivirals to mitigate human infections. Pursuing our search for new bioactive plant-derived molecules, we study several diterpene derivatives synthesized from jatropholones A and B and carnosic acid isolated from Jatropha isabellei and Rosmarinus officinalis, respectively. Here, we investigate the antiviral effect of the diterpenes against human adenovirus (HAdV-5) that causes several infections for which there is no approved antiviral therapy yet. Ten compounds are evaluated and none of them present cytotoxicity in A549 cells. Only compounds 2, 5 and 9 inhibit HAdV-5 replication in a concentration-dependent manner, without virucidal activity, whereas the antiviral action takes place after virus internalization. The expression of viral proteins E1A and Hexon is strongly inhibited by compounds 2 and 5 and, in a lesser degree, by compound 9. Since compounds 2, 5 and 9 prevent ERK activation, they might exert their antiviral action by interfering in the host cell functions required for virus replication. Besides, the compounds have an anti-inflammatory profile since they significantly inhibit the levels of IL-6 and IL-8 produced by THP-1 cells infected with HAdV-5 or with an adenoviral vector. In conclusion, diterpenes 2, 5 and 9 not only exert antiviral activity against adenovirus but also are able to restrain pro-inflammatory cytokines induced by the virus.

A paraguayan toad Rhinella schneideri preparation based on Mbya tradition increases mitochondrial bioenergetics with migrastatic effects dependent on AMPK in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In Paraguay, healers from the Mbya culture treat cancer with a recipe prepared with the native toad Rhinella schneideri. However, the chemical composition and biological effects of the recipe remain unknown. AIM OF THE STUDY: The aim is to determine the composition of the traditional preparation made using the toad R. schneideri and to evaluate its effect on human breast cancer (BC) cells. MATERIALS AND METHODS: The metabolites contained in the preparation were concentrated using XAD-7 resin, and the concentrate was analyzed by HPLC-MS/MS. The effect of the preparation was assessed in normal (MCF10F) and BC cells (MDA-MB-231 and MCF7). The mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) levels, and cell cycle progression were determined by flow cytometry. The oxygen consumption rate (OCR) was measured by Clark electrode, and fibronectin-dependent migration in normoxia and hypoxia-like conditions were evaluated by transwell assay. RESULTS: From the Amberlite-retained extract from the preparation, 24 compounds were identified, including alkaloids, amino acids, bufadienolides, and flavonoids, among others. The crude extract (CE) did not affect cell cycle progression and viability of BC cell lines. Moreover, it did not make cancer cells more sensitive to the cytotoxic effect of the chemotherapeutics doxorubicin and teniposide. On the other hand, the CE reduced the menadione-induced ROS production and increased NADH, Δψm, and the OCR. Respiratory complexes I and III as well as ATP synthase levels were increased in an AMPK-dependent manner. Moreover, the CE inhibited the migration of BC cells in normoxia and a hypoxia-like condition using CoCl2 as a HIF1α-stabilizing agent. This latter effect involved an AMPK-dependent reduction of HIF1α levels. CONCLUSIONS: The Paraguayan toad recipe contains metabolites from the toad ingredient, including alkaloids and bufadienolide derivatives. The CE lacks cytotoxic effects alone or in combination with chemotherapeutics. However, it increases mitochondrial bioenergetics and inhibits the cancer cell migration in an AMPK-dependent manner in BC cells. This is the first report of the in vitro anticancer effect of a traditional Rhinella sp. toad preparation based on Mbya tradition.

Phenolic Composition and α-Glucosidase Inhibition of Leaves from Chilean Bean Landraces.

The MeOH:H2O (7:3) extracts of leaves from Chilean bean landraces were assessed for total phenolic (TP), total flavonoid (TF), total proanthocyanidin (TPA) content, antioxidant capacity (ORAC, FRAP, TEAC, CUPRAC, DPPH) and the inhibition of enzymes associated with metabolic syndrome (α-glucosidase, α-amylase, pancreatic lipase). The chemical profiles were analyzed by HPLC-DAD. Higher antioxidant activity in the ORAC and CUPRAC assay was found for the landrace Coscorrón, and the best effect in the TEAC for Sapito, respectively. The main phenolics were flavonol glycosides and caffeic acid derivatives. The extracts presented strong activity against α-glucosidase, but were inactive towards α-amylase and pancreatic lipase. The leaf extract from the Sapito landrace was fractionated to isolate the main α-glucosidase inhibitors, leading to caffeoylmalic acid with an IC50 of 0.21 µg/mL. The HPLC fingerprints of the leaves differentiate three groups of chemical profiles, according to the main phenolic content. A significant correlation was found between the α-glucosidase inhibition, the content of caffeoylmalic acid (r = -0.979) and kaempferol 3-O-ß-D-glucoside (r = 0.942) in the extracts. The presence of α-glucosidase inhibitors in the leaves of Chilean beans support their potential as a source of bioactive compounds.

The use of medicinal plants by Paraguayan migrants in the Atlantic Forest of Misiones, Argentina, is based on Guaraní tradition, colonial and current plant knowledge.

ETHNOPHARMACOLOGICAL RELEVANCE: Information on the use of medicinal plants in the daily life by Paraguayan people is scarce in mainstream scientific literature. The study on the Paraguayan diaspora in the Provincia de Misiones, Argentina, gives an insight into Guaraní traditions, colonial legacy and current search for new medicinal plants to address new health challenges. AIM OF THE STUDY: To document the use of medicinal plants by Paraguayan mestizo migrants who moved into a new country, yet within the same ecological region. The present and past uses of medicinal plants were compared to understand the continuity and change in the Paraguayan herbal pharmacopoeia. MATERIALS AND METHODS: Fieldwork based on ethnographic and ethnobotanical techniques was carried out in the Provincia de Misiones, Argentina, in 2014, 2015, and 2019. Eighty-five Paraguayan migrants and their descendants from eastern Paraguay took part in the study. The list of recorded plants was compared with the information in historical sources from Paraguay, to examine the continuity and changes in Paraguayan herbal medicine, and with the present-day ethnobotanical studies from Paraguay. Ethnopharmacological and phytochemical studies on the medicinal plants with the highest consensus of uses were reviewed. RESULTS: Altogether, 204 medicinal plant species were recorded. The most frequently mentioned species represented a combination of plants native to the New and Old World. Nearly 40% of the present-day Paraguayan pharmacopoeia shows continuity from colonial and post-colonial periods. Plants were used for 19 medical categories, of which digestive, circulatory and those belonging to humoral medicine were the most prevalent. The ongoing search of plants to treat new health problems is illustrated by reports of 40 species used for hypertension, 26 for diabetes and 18 to lower cholesterol. There is still little evidence for the effectiveness of these plants in the pharmacological literature. Paraguayan migrants were able to continue their traditional plant medicine in Misiones, Argentina, in a substantial way. CONCLUSION: This study was carried out in a geographic area with a long-standing tradition of Guaraní medicine. Paraguayan migrants in Misiones integrate pre-Hispanic Guaraní names and uses of plants and old humoral concepts with current adaptation of plants to meet new health challenges. Several of the uses described in early colonial times are still practiced, giving a solid background for in-depth studies of the local pharmacopoeia.

Iridoids and polyphenols from chilean Gaultheria spp. berries decrease the glucose uptake in Caco-2 cells after simulated gastrointestinal digestion.

Berries are rich food sources of potentially health-beneficial (poly)phenols. However, they may undergo chemical modifications during gastrointestinal digestion. The effect of simulated gastrointestinal digestion on the content and composition of secondary metabolites from Gaultheria phillyreifolia and G. poeppigii berries was studied. The influence of the digested extracts on the in vitro metabolism and absorption of carbohydrates was evaluated. After simulated digestion, 31 compounds were detected by UHPLC-DAD-MS. The total content of anthocyanins decreased by 98-100%, flavonols by 44-56%, phenylpropanoids by 49-75% and iridoids by 33-45%, the latter showing the highest stability during digestion. Digested extracts inhibited α-glucosidase (IC50 2.8-24.9 µg/mL) and decreased the glucose uptake in Caco-2 cells by 17-28%. Moreover, a decrease in the mRNA expression of glucose transporters SGLT1 (38-92%), GLUT2 (45-96%), GLUT5 (28-89%) and the enzyme sucrase-isomaltase (82-97%) was observed. These results show the effect of simulated gastrointestinal digestion on the content and composition of Gaultheria berries.

Antiglycating Effect of Phenolics from the Chilean Currant Ribes cucullatum under Thermal Treatment.

Numerous dietary polyphenols possess antiglicating activity, but the effects of thermal treatment on this activity are mostly unknown. The effect of thermal treatment in the antiglycating activity of polyphenolic enriched extracts (PEEs) from Ribes cucullatum towards glyoxal-induced glycation of sarcoplasmic proteins was assessed. Sarcoplasmic proteins from chicken, beef, salmon, and turkey, were incubated 2 h at 60 °C with and without glyoxal and different concentrations of PEEs (0.25, 0.5, 1, and 5 mg/mL). The antiglycating activity was evaluated by: (1) Lys and Arg consumption, (2) Carboxymethyl lysine (CML) generation, and (3) lipid-derived electrophiles inhibition in a gastric digestion model. Protective effects were observed against CML generation in proteins and a decrease of electrophiles in the gastric digestion model. A dose-dependent consumption of Lys and Arg in proteins/PEEs samples, indicated the possible occurrence of quinoproteins generation from the phenolics. Protein/PEEs incubations were assessed by: (1) High pressure liquid chromatography analysis, (2) Gel electrophoresis (SDS-PAGE), and (3) Redox cycling staining of quinoproteins. Protein/PEEs incubations produced: (1) Decrease in phenolics, (2) increase of protein crosslinking, and (3) dose-dependent generation of quinoproteins. We demonstrate that phenolic compounds from R. cucullatum under thermal treatment act as antiglycating agents, but oxidative reactions occurs at high concentrations, generating protein crosslinking and quinoproteins.

Phenolics from the Bolivian highlands food plant Ombrophytum subterraneum (Aspl.) B. Hansen (Balanophoraceae): Antioxidant and α-glucosidase inhibitory activity.

The Balanophoraceae Ombrophytum subterraneum is an endemic highland food plant occurring in Bolivia, northern Argentina and Chile. The upper part of this parasitic plant is eaten fresh. The aim of this work was to characterize the compounds occurring in the edible part and to assess any differences between the inflorescence and tuber. Ethanol extracts of the different plant parts were analyzed for antioxidant activity by the ORAC, TEAC, FRAP, CUPRAC and DPPH methods as well as for the effect on enzymes related to metabolic syndrome (α-amylase, α-glucosidase and pancreatic lipase). The main constituents were isolated by a combination of gel permeation in Sephadex LH-20 and countercurrent chromatography (CCC). Five compounds were isolated and fully identified by spectroscopic and spectrometric means. The new 3',5,5',7-tetrahydroxyflavanone 7-O-ß-D-1 â†’ 6 diglucoside was isolated for the first time. HPLC-ESI-MS/MS analysis allowed the identification of 19 compounds, including flavanones, flavanols, flavonols, dehydroflavonols and lignans, mainly as glycosides. A strong inhibition towards α-glucosidase was observed for the edible parts (IC50: 1.46 µg/mL) as well as for the tuber and inflorescence (IC50: 1.56 µg/mL and 0.87 µg/mL, respectively). A significant correlation was established between the total phenolic and the antioxidant capacity of the extracts as well as with the content of the new flavanone diglucoside. This is the first comprehensive report on the naturally occurring antioxidants and enzyme inhibitors from this native highland food resource.

The Parotoid Gland Secretion from Peruvian Toad Rhinella horribilis (Wiegmann, 1833): Chemical Composition and Effect on the Proliferation and Migration of Lung Cancer Cells.

Since Rhinella sp. toads produce bioactive substances, some species have been used in traditional medicine and magical practices by ancient cultures in Peru. During several decades, the Rhinella horribilis toad was confused with the invasive toad Rhinella marina, a species documented with extensive toxinological studies. In contrast, the chemical composition and biological effects of the parotoid gland secretions (PGS) remain still unknown for R. horribilis. In this work, we determine for the first time 55 compounds from the PGS of R. horribilis, which were identified using HPLC-MS/MS. The crude extract inhibited the proliferation of A549 cancer cells with IC50 values of 0.031 ± 0.007 and 0.015 ± 0.001 µg/mL at 24 and 48 h of exposure, respectively. Moreover, it inhibited the clonogenic capacity, increased ROS levels, and prevented the etoposide-induced apoptosis, suggesting that the effect of R. horribilis poison secretion was by cell cycle blocking before of G2/M-phase checkpoint. Fraction B was the most active and strongly inhibited cancer cell migration. Our results indicate that the PGS of R. horribilis are composed of alkaloids, bufadienolides, and argininyl diacids derivatives, inhibiting the proliferation and migration of A549 cells.

Isolation and characterization of secondary metabolites from Gaultheria tenuifolia berries.

Gaultheria berries (Ericaceae) are consumed as food or used in folk medicine throughout the world. In the present study, Gaultheria tenuifolia berries were studied to describe their polyphenol and iridoid composition, aroma volatiles, and cytoprotective effects. In total, 14 metabolites were isolated using a combination of countercurrent chromatography and Sephadex LH-20, namely, cyanidin-3-O-ß-galactoside, cyanidin-3-O-ß-arabinoside, 3-O-caffeoylquinic acid, 5-O-caffeoylshikimic acid, quercetin, quercetin-3-O-ß-glucuronide, quercetin-3-O-ß-rutinoside, quercetin-3-O-ß-glucoside, quercetin-3-O-ß-arabinoside, quercetin-3-O-ß-rhamnoside, 6α-hydroxydihydromonotropein-10-trans-cinnamate, monotropein-10-trans-cinnamate, and an (epi)-catechin dimer and trimer. Other flavan-3-ols, proanthocyanidins, and iridoids were tentatively identified by spectroscopic and spectrometric means in the fruit extracts. The tentative volatile organic compound characterization pointed to methyl salicylate as responsible for the aroma of this species. The extracts showed significant cytoprotective effects in an oxidative stress model in human gastric epithelial cells. This is the first report on the isolation, characterization, and potential biological activity of secondary metabolites from G. tenuifolia berries and insights on its possible application as a functional food. PRACTICAL APPLICATION: Berries are desirable fruit species because of their phytochemical composition and pleasant taste. Gaultheria berries are special due to their high content of iridoids and the presence of salicylic acid derivatives. Aroma of native berries is relevant for the development of new products reflecting the local identity and use of fruits. The present work involves cooperation of academia and industry on the constituents of the native products. The results provided in this article could be useful for the introduction of this species in the food and nutraceutical industries.

Effects of gastrointestinal digested polyphenolic enriched extracts of Chilean currants (Ribes magellanicum and Ribes punctatum) on in vitro fecal microbiota.

Chilean currants (Ribes magellanicum and Ribes punctatum) are wild polyphenol-rich berries with interesting bioactivities in several in vitro models. The aim of this study was to investigate the effects of the pre-digested PEE (polyphenol-enriched extract) in a simulated colon model. Fruits were extracted, submitted to simulated gastrointestinal digestion and further colonic fermentation with feces from healthy human donors. Samples were taken at 1, 4, 8 and 24 h of incubation, monitoring pH, ammonia, branched-chain fatty acids (BCFA), short-chain fatty acids (SCFA) and bacterial growth. FOS (fructooligosaccharides) and fecal slurry without treatments were positive and negative control, respectively. Both Ribes species reduced (p < 0.05) both BCFA and SCFA at 24 h. R. punctatum promoted the growth (p < 0.05) of beneficial bacteria such as Clostridium cluster XIVa, and Faecalibacterium prausnitzii; while a trend to increase Akkermansia muciniphila was observed. R. magellanicum increased (p < 0.05) Clostridium cluster XIVa population. Escherichia coli, Lactobacillus spp. and Bifidobacterium spp. remained unaffected. Our results suggest that polyphenols from R. punctatum and R. magellanicum may modulate both bacterial metabolism and some selected gut beneficial bacteria under simulated conditions. Therefore, Chilean currants might be useful as supplements to maintain a healthy colon; however, further in vivo studies are needed to confirm their effect and their mechanisms.

Integral use of Argentinean Solanum betaceum red fruits as functional food ingredient to prevent metabolic syndrome: effect of in vitro simulated gastroduodenal digestion.

The "red chilto" (Solanum betaceum Cav) is a native fruit from the Yungas forest of Argentina. Red chilto is a neglected and underutilized native species (NUS). The objective of this work was to evaluate the potentiality of pulp, seed and skin from "red chilto" as a functional food ingredient to add value to a native resource of Argentine Yungas to promote sustainable integral use of it. The powders have low carbohydrate and sodium content and are a source of vitamin C, phenolic acids (rosmarinic acid and caffeoylquinic acid), anthocyanins, condensed tannins, carotenoids, potassium, and fiber. The phenolics of chilto powders showed, before and after simulated gastroduodenal digestion, antioxidant activity (ABTS•+; H2O2; O2 •; HO•) and were able to inhibit enzymes related to metabolic syndrome, such as α-glucosidase, α-amylase and lipase. Chilto powder showed hypoglycemic effect by increasing glucose adsorption, decreasing glucose diffusion rate and by promoting glucose transport across the cell membrane. These results suggest the potential of Argentinean "red chilto" fruits as functional food ingredients or dietary supplements to prevent metabolic syndrome principally by its antioxidant, hypoglycemic and hypolipemic effects.

Iridoids and Amino Acid Derivatives from the Paraguayan Crude Drug Adenocalymma marginatum (ysypó hû).

The crude drug ysypó hû (Adenocalymma marginatum DC., Bignoniaceae) is used traditionally by the Guarani of Eastern Paraguayan as a male sexual enhancer. The aim of the present study was to identify the main constituents of the crude drug and to evaluate the in vitro inhibitory activity towards the enzyme phosphodiesterase-5 (PDE-5). The main compounds were isolated by counter-current chromatography (CCC). The metabolites were identified by spectroscopic and spectrometric means. The chemical profiling of the extracts was assessed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). The crude extract and main isolated compounds were tested for their PDE-5 inhibitory activity using commercial kits. The iridoid theviridoside and 4-hydroxy-1-methylproline were isolated as the main constituent of the crude drug. Four chlortheviridoside hexoside derivatives were detected for the first time as natural products. Chemical profiling by HPLC-MS/MS led to the tentative identification of nine iridoids, six phenolics, and five amino acids. The crude extracts and main compounds were inactive towards PDE-5 at concentrations up to 500 µg/mL. Iridoids and amino acid derivatives were the main compounds occurring in the Paraguayan crude drug. The potential of ysypó hû as a male sexual enhancer cannot be discarded, since other mechanisms may be involved.

Bioactive Constituents from South American Prosopis and their Use and Toxicity.

BACKGROUND: The pods from several South American Prosopis species have been considered relevant food in arid and semi-arid South America since prehistoric times. Traditionally the meal from the pods was processed to prepare different foods and beverages. OBJECTIVE: The objective was to discuss literature from the archaeological evidence of use to study the chemistry and (bio)activity of the extracts and secondary metabolites occurring in different Prosopis food products. METHODS: The review was carried out by searching electronic databases, including ScienceDirect, SciFinder, Scopus, Scielo, Google Scholar, PubMed and hand-search on literature. The review mainly covers studies performed in the year 1995-2019 and the first-hand experience of the authors. References on the historical and prehistorical uses of the natural resource were also included. RESULTS: In the last decades, most studies on the edible South American Prosopis focused on the constituents of pods meal, traditional preparations and by-products. Total 45 flavonoids, ellagic acid derivatives, catechin and simple phenolics were identified. Alkaloids occur mainly in the leaves, that are not used for human nutrition but as food for domestic animals. Piperidine alkaloids, tryptamine, tyramine and ß-phenethylamine were isolated and identified from several species. The (bio)activity studies included mainly the antioxidant effect, antiinflammatory and enzyme inhibition associated with metabolic syndrome. The products showed no toxicity or mutagenic effect. CONCLUSION: While data on the chemistry, some (bio)activities and toxicity are available for the pods meal and byproducts, little is known about the composition of the fermented Algarrobo beverages. Further studies are needed on the digestion of Algarrobo products both in humans and cattle.

Andean Prumnopitys Andina (Podocarpacae) Fruit Extracts: Characterization of Secondary Metabolites and Potential Cytoprotective Effect.

The fruits from the Chilean Podocarpaceae Prumnopitys andina have been consumed since pre-Hispanic times. Little is known about the composition and biological properties of this fruit. The aim of this work was to identify the secondary metabolites of the edible part of P. andina fruits and to assess their antioxidant activity by means of chemical and cell-based assays. Methanol extracts from P. andina fruits were fractionated on a XAD7 resin and the main compounds were isolated by chromatographic means. Antioxidant activity was determined by means of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), ferric reducing power (FRAP), trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The cytoprotective activity of the extract against oxidative and dicarbonyl stress was evaluated in human gastric epithelial cells (AGS). The total intracellular antioxidant activity (TAA) of the extract was determined in AGS cells. The inhibition of meat lipoperoxidation was evaluated under simulated gastric digestion conditions. Rutin, caffeic acid ß-glucoside and 20-hydroxyecdysone were identified as major components of the fruit extract. Additional compounds were identified by high-performance liquid chromatography diode-array detector mass spectrometry (HPLC-DAD-MSn) and/or co-injection with standards. Extracts showed dose-dependent cytoprotective effects against oxidative and dicarbonyl-induced damage in AGS cells. The TAA increased with the pre-incubation of AGS cells with the extract. This is the first report on the composition and biological activity of this Andean fruit.

Polyphenol Composition and (Bio)Activity of Berberis Species and Wild Strawberry from the Argentinean Patagonia.

The Argentinean Patagonia berries Berberis microphylla, Berberis darwinii, and Fragaria chiloensis ssp. chiloensis f. patagonica were investigated for their polyphenol content and composition by means of liquid chromatography coupled to diode array detection and electrospray ionization tandem mass spectrometry. The in vitro antioxidant activity and inhibition of metabolic syndrome-associated enzymes (α-glucosidase, α-amylase, and lipase) of the fruit extracts was assessed. The most complex polyphenol profile was found in the Berberis samples, with 10 anthocyanins, 27 hydroxycinnamic acids, 3 proanthocyanidins, 2 flavan-3-ol, and 22 flavonols. Fragaria presented four anthocyanins, nine ellagitannins, two proanthocyanidin dimers, one flavan-3-ol, and five flavonols. The Berberis samples showed the best antioxidant capacity, while Fragaria displayed better activity against α-glucosidase and lipase. The phenolic content and composition of the Argentinean Patagonia berries was similar to that reported for Chilean samples but with some chemical differences between Eastern (Argentina) and Western (Chile) Patagonia. The data obtained supports the consumption of these berries as sources of beneficial polyphenols.

Effect of simulated gastrointestinal digestion on polyphenols and bioactivity of the native Chilean red strawberry (Fragaria chiloensis ssp. chiloensis f. patagonica).

The native Chilean red strawberry (Fragaria chiloensis spp. chiloensis f. patagonica) is a wild strawberry with high polyphenol content and antioxidant activity originating in central-southern Chile. The aim of the present work was to compare the composition and bioactivity of polyphenol-concentrated extracts (PCE) of the fruit, before and after simulated gastrointestinal digestion (GID). Twenty nine compounds were tentatively identified in the non-digested PCE. After GID, 26 and 23 compounds were detected, in the gastric and intestinal steps, respectively. The compounds that were more affected by the simulated GID were cyanidin hexoside, bis hexahydroxydiphenic acid (HHDP) hexosides, bis HHDP galloyl hexosides, apigenin hexoside, and quercetin dihexoside. Results show a decrease in the total phenolic content by 3.4% and 43% at the end of the gastric and intestinal steps, respectively. In the same way, the total flavonoid content decreased by 60.4% and 90.9% at the end of the gastric and intestinal step, respectively. Overall, the antioxidant activity decreased during the gastrointestinal process; as well as the inhibitory activity against α-glucosidase and lipase was reduced by the simulated digestion. These results are a first approach to understand the effects induced by the gastrointestinal digestion on the bioactivity and polyphenolic profile of this native fruit.

Cytotoxicity and antimitotic activity of Rhinella schneideri and Rhinella marina venoms.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhinella schneideri and Rhinella marina are toad venoms distributed in different parts of the world, including Brazil, Columbia and amazon. Venoms extracted from different species have many clinical applications such as antimicrobial cardiotonics and treatment of cancer. Aim of the study; In this study, we aim to investigate the effect of venoms extracted from R. schneideri and R. marina on cancer cells and verify possible mechanism of action. MATERIAL AND METHOD: Cytotoxicity analyses was performed using the resazurin reduction assay, where different concentrations of venoms were tested against sensitive CCRF-CEM and P-gp overexpressing ADR/CEM5000 leukemia cells. Programmed cell death was investigated using the flow cytometric annexin V/propidium iodide apoptosis assay. Furthermore, we analyzed flow cytometric cell cycle analyses of CCRF-CEM cells. Effect on tubulin formation was tested using molecular docking and fluorescence microscopy of U2OS-GFP-α-tubulin osteosarcoma cells treated for 24 h with venoms. RESULTS: Cytotoxicity assays revealed a strong activity towards wild-type CCRF-CEM cells (IC50 values of 0.202 ±â€¯0.005 µg/ml and 0.18 ±â€¯0.007 µg/ml for R. schneideri and R. marina, respectively) and multidrug-resistant CEM/ADR5000 cells (IC50 0.403 ±â€¯0.084 µg/ml and 0.32 ±â€¯0.077 µg/ml for R. schneideri and R. marina, respectively). The venoms induced apoptosis as major mechanism of cell death. The venoms induced strong G2/M cell arrest in CCRF-CEM cells. We suggested tubulin as a major target for the venoms. In silico molecular docking of the major constituents of the venoms, i.e. bufalin, marinobufagin, telocinbufagin, hellebrigenin, showed strong binding affinities to tubulin. This result was verified in vitro. The venoms dysregulated microtubule arrangement of U2OS cells expressing GFP-labeled tubulin. Toxicity predictions by QSAR methodology highlighted the toxic features of bufadienolides. CONCLUSION: Our study demonstrated the importance of toad venoms as source of cytotoxic compounds that may serve as lead compounds for the development of novel anticancer drugs.

Patagonian berries as native food and medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Patagonia is the southernmost part of the South American continent including Chile and Argentina. Berries and wild fruits have been gathered by the native Patagonians as food and medicine for over 14,000 years. The economic potential of the native berries as health promoting and relevant sources of bioactive substances has become apparent with several studies in the last decades. AIM OF STUDY: This work aims to provide an insight into the ethnohistorical records of wild edible fruits from Patagonia starting with the archeobotanical studies to the contemporary use of the resources. The chemical and bioactivity studies on the native fruits are presented and discussed. METHODOLOGY: A search of electronic databases including Scopus, Scielo, Google Scholar, PubMed, ScienceDirect and SciFinder, as well as hand-search was carried out to perform an integrative review on the native Patagonian berries. RESULTS: The use of native berries as food and medicine by the ancient hunter-gatherer societies can be traced back to the early occupation of Patagonia. The same species used in prehistoric times are still used as food by the contemporary population in this area. Chemical and bioactivity studies have reported remarkable activities in several of the native berries, including calafate (Berberis spp.), native strawberry (Fragaria chiloensis), currants (Ribes spp.), Patagonian raspberries (Rubus spp.) and maqui (Aristotelia chilensis) fruits. The increasing demand for maqui and calafate led to the selection of varieties for commercial production. The fruit constituents show strong antioxidant and inhibitory effect towards enzymes associated with metabolic syndrome, including α-amylase, α-glucosidase and lipase. Some berry constituents exert anti-inflammatory effects in vitro. The phytochemicals identified include a wide array of phenolics of different structural skeletons. Changes in composition and bioactivity after simulated gastric and intestinal digestion, as well as colonic fermentation, have been reported in some Patagonian species. CONCLUSIONS: Patagonian berries are a relevant source of bioactive compounds with several health promoting properties. The long tradition of use and the interest of the population for their consumption has led to the development of some of this fruits as new potential crops. The ethnobotanical evidence shows a shared knowledge among the different indigenous communities on plant uses according to the local resources, and an integration of the ancient knowledge into the contemporary society. Other species are being investigated to get a more complete picture of the food and medicinal plants from Patagonia.

Antioxidant activity and the isolation of polyphenols and new iridoids from Chilean Gaultheria phillyreifolia and G. poeppigii berries.

The berries from the native Chilean Gaultheria phillyreifolia and G. poeppigii are appreciated for their sweet taste and aroma. Fruits from both species were investigated for their secondary metabolite composition and antioxidant activity. The extracts were submitted to membrane chromatography to separate anthocyanins from copigments. Four anthocyanins were isolated by counter-current chromatography (CCC) and identified as cyanidin galactoside, cyanidin arabinoside, delphinidin galactoside and delphinidin arabinoside. From the copigments, CCC allowed the separation of quercetin(Q)-3-arabinoside, Q-3-rutinoside Q-3-rhamnoside and 3-caffeoylquinic acid. Additionally, the iridoids monotropein-10-trans-coumarate, monotropein-10-trans-cinnamate and 6α-hydroxy-dihydromonotropein-10-trans-cinnamate were isolated. The latter two iridoids are reported here for the first time. Some 34 other compounds were tentatively identified by HPLC-DAD-ESI-MSn. The antioxidant activity showed differences between anthocyanins and copigments from both species. Main compounds were quantified and submitted to a Partial-Least Square Discriminant Analysis (PLS-DA). This is the first report on the isolation of phytochemicals from the selected Chilean Gaultheria species.

Male sexual enhancers from the Peruvian Amazon.

ETHNOPHARMACOLOGICAL RELEVANCE: Selected Peruvian Amazon plants are macerated into sugar cane distillates to prepare alcoholic beverages used to improve male sexual performance. The tree bark from Campsiandra angustifolia Spruce ex Benth (Fabaceae), Swartzia polyphylla DC (Fabaceae), Minquartia guianensis Aubl. (Olacaceae) and Thynantus panurensis (Bureau) Sandwith (Bignoniaceae) usually are used as crude drugs in mixtures of several ingredients. AIM OF STUDY: Describe the chemical composition of the most traded traditional male enhancer beverages, namely "Levántate Lazaro" and "Siete veces sin sacarla", and their single crude drug constituents, as well as their inhibitory activity towards the enzyme phosphodiesterase-5. The presence of pro-sexual drugs such as Sildenafil® and derivatives was assessed in the samples. MATERIALS AND METHODS: Single plant constituents and the preparation mixtures were purchased in the Mercado Belen (Iquitos, Peru). Chemical profiling was carried out by HPLC-DAD-ESI-MS/MS. The extracts were assessed for phosphodiesterase-5 inhibition. The occurrence of pro-sexual drugs was determined by HPLC-DAD-ESI-MS/MS. RESULTS: Chemical profiling allowed the identification of condensed tannins as the main constituents of C. angustifolia and S. polyphylla, hydrolysable tannins for M. guianensis, and C-glycosides for T. panurensis. The traditional preparations showed similar composition compared to the crude drugs. At 200 µg/mL, the traditional preparation "Levántate Lázaro" and "Siete veces sin sacarla" inhibited the phosphodiesterase-5 by 49.88% and 27.90%, respectively. No adulterations with pro-sexual drugs were found in the samples. From the crude drugs, low effect was found for the extracts of S. polyphylla and T. panurensis and high activity for C. angustifolia which inhibited the enzyme by 89.37% and 81.32% at 200 and 100 µg/mL, respectively. CONCLUSION: The traditional preparations used to improve sexual performance in the Peruvian Amazon showed activity as phosphodiesterase-5 inhibitors. The most active ingredient of the traditional preparations was C. angustifolia, with some contribution from T. panurensis. These results encourage additional studies, including animal models to confirm the male enhancer effect of the preparations.