Acute Administration of Calafate (Berberis microphylla) Extract Induces the Expression of Thermogenic Markers and Modulates Gut Microbiota in Mice Fed a High-Fat Chow Diet.

INTRODUCTION: Obesity, characterized by excess adipose tissue, is a major public health problem worldwide. Brown adipose tissue (BAT) and beige adipose tissue participate in thermogenesis through uncoupling protein 1 (UCP1). Polyphenols including those from Calafate (a native polyphenol-rich Patagonian berry), are considered as potential anti-obesity compounds due to their pro-thermogenic characteristics. However, polyphenols are mainly metabolized by the gut microbiota (GM) that may influence their bioactivity and bioavailability. The aim of this study was to determine the impact of dietary administration with a Calafate polyphenol-rich extract on thermogenic activity of BAT and beige adipose tissue and GM composition. METHODS: Eight-week-old C57BL6 mice (n = 30) were divided into 4 groups to receive for 24 weeks a control diet (C), a high-fat diet alone (HF), or high-fat diet supplemented with Calafate extract (HFC) or the same high-fat diet supplemented with Calafate extract but treated with antibiotics (HFCAB) from week 19-20. Administration with Calafate extract (50 mg/kg per day) was carried out for 3 weeks from week 21-23 in the HFC and HFCAB groups. After euthanasia, gene expression of thermogenic markers was analyzed in BAT and inguinal white adipose tissue (iWAT). Transmission electron microscopy was performed to assess mitochondrial morphology and cristae density in BAT. GM diversity and composition were characterized by deep sequencing with the MiSeq Illumina platform. RESULTS: Calafate extract administration had no effect on weight gain in mice fed a high-fat diet. However, it prevented alterations in mitochondrial cristae induced by HFD and increased Dio2 expression in BAT and iWAT. The intervention also influenced the GM composition, preventing changes in specific bacterial taxa induced by the high-fat diet. However, the antibiotic treatment prevented in part these effects, suggesting the implications of GM. CONCLUSION: These results suggest that the acute administration of a Calafate extract modulates the expression of thermogenic markers, prevents alterations in mitochondrial cristae and intestinal microbiota in preclinical models. The study highlights the complex interaction between polyphenols, thermogenesis, and the GM, providing valuable insights into their potential roles in the treatment of obesity-related metabolic diseases.

Berberis microphylla G. Forst Intake Reduces the Cardiovascular Disease Plasmatic Markers Associated with a High-Fat Diet in a Mice Model.

Polyphenols are bioactive substances that participate in the prevention of chronic illnesses. High content has been described in Berberis microphylla G. Forst (calafate), a wild berry extensively distributed in Chilean-Argentine Patagonia. We evaluated its beneficial effect through the study of mouse plasma metabolome changes after chronic consumption of this fruit. Characterized calafate extract was administered in water, for four months, to a group of mice fed with a high-fat diet and compared with a control diet. Metabolome changes were studied using UHPLC-DAD-QTOF-based untargeted metabolomics. The study was complemented by the analysis of protein biomarkers determined using Luminex technology, and quantification of OH radicals by electron paramagnetic resonance spectroscopy. Thirteen features were identified with a maximum annotation level-A, revealing an increase in succinic acid, activation of tricarboxylic acid and reduction of carnitine accumulation. Changes in plasma biomarkers were related to inflammation and cardiovascular disease, with changes in thrombomodulin (-24%), adiponectin (+68%), sE-selectin (-34%), sICAM-1 (-24%) and proMMP-9 (-31%) levels. The production of OH radicals in plasma was reduced after calafate intake (-17%), especially for the group fed with a high-fat diet. These changes could be associated with protection against atherosclerosis due to calafate consumption, which is discussed from a holistic and integrative point of view.

Sequential Biorefining of Bioactive Compounds of High Functional Value from Calafate Pomace (Berberis microphylla) Using Supercritical CO2 and Pressurized Liquids.

A biorefinery process was developed for a freeze-dried pomace of calafate berries (Berberis microphylla). The process consisted of extraction of lipophilic components with supercritical CO2 (scCO2) and subsequent extraction of the residue with a pressurized mixture of ethanol/water (1:1 v/v). scCO2 extracted oil from the pomace, while pressurized liquid extraction generated a crude extract rich in phenols and a residue rich in fiber, proteins and minerals. Response surface analysis of scCO2 extraction suggested optimal conditions of 60 °C, 358.5 bar and 144.6 min to obtain a lipid extract yield of 11.15% (d.w.). The dark yellow oil extract contained a good ratio of ω6/ω3 fatty acids (1:1.2), provitamin E tocopherols (406.6 mg/kg), and a peroxide index of 8.6 meq O2/kg. Pressurized liquid extraction generated a polar extract with good phenolic content (33 mg gallic acid equivalents /g d.w.), anthocyanins (8 mg/g) and antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl test = 25 µg/mL and antioxidant activity = 63 µM Te/g). The extraction kinetics of oil by scCO2 and phenolic compounds were optimally adjusted to the spline model (R2 = 0.989 and R2 = 0.999, respectively). The solid extracted residue presented a fiber content close to cereals (56.4% d.w.) and acceptable values of proteins (29.6% d.w.) and minerals (14.1% d.w.). These eco-friendly processes valorize calafate pomace as a source of ingredients for formulation of healthy foods, nutraceuticals and nutritional supplements.

Efecto de la suplementación del medio de incubación con extracto de Berberis microphylla (calafate) sobre la funcionalidad espermática en semen bovino descongelado / Effect of supplementation of incubation medium with Berberis microphylla (calafate) extract on sperm functionality in thawed bovine semen

RESUMEN: En el semen criopreservado, los procesos de congelación/descongelación y posterior manipulación, dañan las células espermáticas provocando disminución de la capacidad fecundante de los espermatozoides descongelados. Estos procesos han sido asociados con el estado de estrés oxidativo (EO) inducido por altos niveles de especies reactivas de oxígeno (EROS), causando daño a la función y estructura espermática. Los espermatozoides descongelados pueden ser protegidos de este daño, con la adición de antioxidantes (AO) al medio de incubación. El fruto de Calafate (Berberis microphylla G. Forst.) posee una alta capacidad antioxidante, lo que hace interesante investigar el efecto de sus componentes antioxidantes en estos procesos biotecnológicos especialmente postdescongelación. El objetivo de este estudio fue determinar el efecto de la suplementación de extracto liofilizado de fruto de Calafate (ELC), sobre la calidad espermática post-descongelación. Previamente se caracterizó el ELC, determinando la actividad antioxidante y metabolitos como fenoles y antocianinas; posteriormente, espermatozoides de bovino descongelados fueron incubados en un medio base suplementado con diferentes concentraciones de ELC. Post-incubación se evaluó la motilidad progresiva; la viabilidad e integridad de la membrana plasmática (SYBR14- PI) y acrosomal (FITC-PNA/PI) y la peroxidación lipídica (BODIPY) por citometría de flujo. La caracterización de ELC demostró que tanto la actividad antioxidante como los fenoles y antocianinas incrementan concomitante con el aumento de la concentración de ELC. La adición de ELC al medio de incubación, dependiendo de la concentración y tiempo de incubación, sería eficaz en proteger la motilidad, viabilidad e integridad de la membrana plasmática y disminuir la lipoperoxidación en los espermatozoides de bovino descongelados.

SUMMARY: In cryopreserved semen, the freezing/thawing process following of manipulation, damage the sperm cell, decreasing the fertilizing capacity of the thawed sperm; being one of the main factors of this damage the oxidative stress. The sperm once thawed can be protected from this damage, with the addition of antioxidants to the incubation medium. The Calafate fruit (Berberis microphylla G. Forst.) has a high antioxidant capacity, making it an interesting resource for investigating the effect of its antioxidant components on biotechnological processes. The objective of this study was to determine the effect of supplementation of Calafate fruit lyophilized extract (ELC) on sperm quality. The lyophilized extract of the Calafate fruit was characterized, determining the antioxidant activity and metabolites such as phenols and anthocyanins; subsequently, thawed bovine sperm were incubated in a medium supplemented with different concentrations of ELC. Post-incubation, progressive motility was evaluated. By flow cytometry, the viability and integrity of the plasma (SYBR14-PI), and acrosomal (FITC-PNA / PI), as well as lipid peroxidation (BODIPY), was determined. The characterization of Calafate fruits lyophilized extract indicated that antioxidant activity, phenols and anthocyanins increased concomitantly with the increase of dose extract used. The addition of ELC to the incubation medium, depending on the concentration and incubation time, would be effective to protect motility, viability and integrity of the plasma membrane and decreased lipid peroxidation in thawed bovine sperm.

Ethnopharmacology, Phytochemistry and Biological Activities of Native Chilean Plants.

The native flora of Chile has unique characteristics due to the geographical situation of the country, with the vast desert in the North, Patagonia in the South, the Andean Mountains on the east and the Pacific Ocean on the west. This exclusivity is reflected in high concentrations of phytochemicals in the fruits and leaves of its native plants. Some examples are Aristotelia chilensis (Molina), Stuntz (maqui), Berberis microphylla G. Forst. (calafate), Peumus boldus Molina (boldo), Ribes magellanicum Poir. (Magellan currant), Ugni molinae Turcz. (murtilla), Rubus geoides Sm. (miñe miñe), Drimys winteri J.R.Forst. & G.Forst. (canelo), Luma apiculata (DC.) Burret (arrayán) distributed throughout the entire Chilean territory. Some of these Chilean plants have been used for centuries in the country's traditional medicine. The most recent studies of phytochemical characterization of parts of Chilean plants show a wide spectrum of antioxidant compounds, phenolic components, terpenoids and alkaloids, which have shown biological activity in both in vitro and in vivo studies. This manuscript covers the entire Chilean territory characterizing the phytochemical profile and reporting some of its biological properties, focusing mainly on antioxidant, anti-inflammatory, antimicrobial, chemopreventive and cytotoxic activity, and potential against diabetes, metabolic syndrome and gastrointestinal disorders.

Fruit provision from Berberis microphylla shrubs as ecosystem service in Nothofagus forest of Tierra del Fuego.

Berry production is a non-wood product worldwide recognized by its nutritional value and taste, but the most studied species are non-native commercial plants in productive areas, leaving aside native berries. We propose that native berries (Berberis microphylla G.Forst) naturally growing in degradation forests areas could diversify livestock establishment production and complement traditional uses (e.g., livestock). The aims of this work were to 1) environmentally characterize (e.g., soil nutrient content and physical conditions, air conditions and photosynthetically active radiation) in three degraded Nothofagus antarctica Oerst. forest (due to past fires and livestock use) of Tierra del Fuego; and 2) evaluate berries production of B. microphylla in terms of quality production (e.g., fruit number and weigh per shrub) and in terms of quality (individual fruit weight, fruit pulp percentage, and soluble solids content) to assess provisioning ecosystem service of this native shrub in different degraded areas. Studied sites were defined as: 1) Severe soil degradation condition (SEV) (high frequency of horses, bulls and some native guanacos year round, severe soil erosion, and shorter herbaceous layer), 2) Moderate soil degradation condition (MOD) (cattle and guanaco year round, intermediate level of soil erosion and intermediate height of herbaceous layer), and 3) Slight soil degradation condition (SLI) (livestock only during winter, but high frequency of native guanacos, lower soil erosion and taller herbaceous layer). (SEV) had the highest air and soil temperature, least soils nutrients content, highest bulk density, the least soil water content and the poorest fruit production. (MOD) had the highest soil water content and nutrient-rich soils, while (SLI) had the highest relative air humidity and PAR. B. microphylla shrubs grow with similar morphology on the different soil degraded condition. The highest fruit production were at (SLI), however the (SEV) had the highest soluble solids. We conclude that calafate shrubs in degraded Nothofagus forests offer a provisioning ecosystem service through their excellent fruits quality. Livestock farms could diversify their production through native fruits taking advantage of the altered areas occupied by B. microphylla. However, we recommend avoiding intensive livestock use in burned forests since it could lead to an irreversible soil erosion. Proper livestock management in Nothofagus burned forest could keep over the time not only the recognized ecosystem provision services (fruits, meat, wood), but also those of regulation and support that calafate shrubs offer and that make the functionality of the ecosystem.

Quality Attributes of Cryoconcentrated Calafate (Berberis microphylla) Juice during Refrigerated Storage.

This study aimed to evaluate the potential of centrifugal block cryoconcentration (CBCC) at three cycles applied to fresh calafate juice. The fresh juice and cryoconcentrate at each cycle were stored for five weeks at 4 °C and quality attributes were analyzed every 7 days. CBCC had significant effects in the calafate juice, since in the last cycle, the cryoconcentrate reached a high value of total soluble solids (TSS, ≈42 °Brix), with final attractive color, and an increase of approximately 2.5, 5.2, 5.1, 4.0 and 5.3 times in relation to the fresh juice values, for total bioactive compounds (TBC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC), respectively. However, at 35 days under storage, these values decreased by 5%, 13%, 15%, 19%, 24% and 27%, for TSS, TBC, DPPH, ABTS, FRAP and ORAC, respectively. Additionally, until the day 14, the panelists indicated a good acceptability of the reconstituted cryoconcentrate. Therefore, CBCC can be considered a novel and viable technology for the preservation of quality attributes from fresh calafate juice with interesting food applications of the cryoconcentrates due to their high stability during storage time in comparison to the fresh juice.

Argentine Patagonia barberry chemical composition and evaluation of its antioxidant capacity.

An important portion of vitamins, minerals and polyphenols components in human diet are captured from fruit consumption. Argentinean Patagonia Berberis microphylla was characterized with the phenolic content, the proximate composition and the identification and quantification of anthocyanins, not-anthocyanins and proteins. The antioxidant capacity of berberis ethanolic extracts (EB) was determined by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. EB was used to reduce production of reactive substances species (ROS) in zebrafish. EB presented a total polyphenols content of 1,035.03 mg GAE/100 g fresh weight (FW). EB presented an ABTS value of 116.25 ± 17 µmol TE/g FW. EB presented a DPPH value of 137.80 ± 1.90 µmol TE/g FW. EB was able of reducing the ROS in zebrafish. Berberies Protein Isolate (BPI) presented proteins with bands from 15 to 62 kDa. BPI presented an ABTS value of 593.11 ± 8.60 µmol TE/g. The BPI duodenal digest presented a value of 641.07 ± 12.60 µmol TE/g digests. PRACTICAL APPLICATIONS: The practical applications of the present study are to increase scientific knowledge for consumers about the quality and benefits of the consumption of the native fruit (Berberis microphylla) from the Patagonia region of Argentine. This work describes the protein profile of berberies, their digestibility and their antioxidant activity. This study allows to better understand the phytonutrients that make up this fruit. Future studies may identify the peptides present in hydrolyzates. The bio-compounds of this fruit could be used as functional ingredients by the food industry for different purposes.

Patagonian Berries: Healthy Potential and the Path to Becoming Functional Foods.

In recent years, there has been an increasing interest in studying food and its derived ingredients that can provide beneficial effects for human health. These studies are helping to understand the bases of the ancestral use of several natural products, including native fruits as functional foods. As a result, the polyphenol profile and the antioxidant capacity of the extracts obtained from different Patagonian native berries have been described. This review aims to provide valuable information regarding fruit quality, its particular compound profile, and the feasibility of producing functional foods for human consumption to prevent disorders such as metabolic syndrome and cardiovascular diseases. We also discuss attempts concerning the domestication of these species and generating knowledge that strengthens their potential as traditional fruits in the food market and as a natural heritage for future generations. Finally, additional efforts are still necessary to fully understand the potential beneficial effects of the consumption of these berries on human health, the application of suitable technology for postharvest improvement, and the generation of successfully processed foods derived from Patagonian berries.

Potent Vasodilator and Cellular Antioxidant Activity of Endemic Patagonian Calafate Berries (Berberis microphylla) with Nutraceutical Potential.

Hydroalcoholic extracts of Patagonian Calafate berry (Berberis microphylla) contain mono or disaccharide conjugated anthocyanins and flavonols. The Liquid Chromatography-Mass Spectrometry (LC-MS) chemical extract profile identified glycosylated anthocyanidins such as delphinidin-, petunidin- and malvidin-3-glucoside as the major constituents. The predominant flavonols were 3-O substituents quercetin-rutinoside or -rhamnoside. Anthocyanins doubled flavonols in mass (13.1 vs. 6 mg/g extract). Polyphenols vascular actions were examined in the rat arterial mesenteric bed bioassay; extract perfusion elicited concentration-dependent vasodilatation mimicked by conjugated anthocyanins standards. Vascular responses of main glycosylated anthocyanins were endothelium-dependent (p < 0.001) and mediated by NO production (p < 0.05). The anthocyanins antioxidant activity determined in isolated endothelial cells (CAA) showed a reduced redox potential as compared to the extract or quercetin. While in the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, the anthocyanins showed an equivalent quercetin potency, the extract was 15-fold less active, proposing that the anthocyanin-induced vasodilation is not due to an antioxidant mechanism. The extract shows promising commercial nutraceutical potential.

Pharmacokinetics of low molecular weight phenolic compounds in gerbil plasma after the consumption of calafate berry (Berberis microphylla) extract.

Calafate is a berry with high concentration of anthocyanins and hydroxycinnamic acids that grows in South Patagonia. To date, no metabolism studies of phenolic compounds using calafate have been carried out. A calafate extract was characterized by HPLC-DAD-ESI-MS/MS. After extract administration (300 mg/kg), a pharmacokinetic study of phenolic compounds in gerbil plasma was performed by GC-MS/MS. Sixteen phenolic acids increased after intake. Phenylacetic acid derivatives exhibit the highest concentration, while main increase of phenolic catabolites was observed 2 h post-intake. 3-hydroxyphenylacetic and phenylacetic acids increased at 4-8 h post-intake. All catabolites found in gerbil plasma exhibit concentration peaks between 0.1 and 1 µM, however no parental anthocyanins were detected. Establish in vivo plasmatic concentration ranges of phenolic compounds derived from polyphenol consumption following WHO recommendations, plays a key role to carry out future in vitro assays in order to correctly assign biological benefits of calafate berry consumption.