Molecular Insights into the Antistress Potentials of Brazilian Green Propolis Extract and Its Constituent Artepillin C.

Propolis, also known as bee-glue, is a resinous substance produced by honeybees from materials collected from plants they visit. It contains mixtures of wax and bee enzymes and is used by bees as a building material in their hives and by humans for different purposes in traditional healthcare practices. Although the composition of propolis has been shown to depend on its geographic location, climatic zone, and local flora; two largely studied types of propolis: (i) New Zealand and (ii) Brazilian green propolis have been shown to possess Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC) as the main bioactive constituents, respectively. We have earlier reported that CAPE and ARC possess anticancer activities, mediated by abrogation of mortalin-p53 complex and reactivation of p53 tumor suppressor function. Like CAPE, Artepillin C (ARC) and the supercritical extract of green propolis (GPSE) showed potent anticancer activity. In this study, we recruited low doses of GPSE and ARC (that did not affect either cancer cell proliferation or migration) to investigate their antistress potential using in vitro cell based assays. We report that both GPSE and ARC have the capability to disaggregate metal- and heat-induced aggregated proteins. Metal-induced aggregation of GFP was reduced by fourfold in GPSE- as well as ARC-treated cells. Similarly, whereas heat-induced misfolding of luciferase protein showed 80% loss of activity, the cells treated with either GPSE or ARC showed 60-80% recovery. Furthermore, we demonstrate their pro-hypoxia (marked by the upregulation of HIF-1α) and neuro-differentiation (marked by differentiation morphology and upregulation of expression of GFAP, ß-tubulin III, and MAP2). Both GPSE and ARC also offered significant protection against oxidative stress and, hence, may be useful in the treatment of old age-related brain pathologies.

[Colorectal cancer preventive effect of combined administration of phenolic acids and supercritical extracts from Angelica sinensis].

This study aimed to investigate the colorectal cancer preventive effect of the combined administration of phenolic acids and supercritical extracts from Angelica sinensis. The AOM/DSS model in mice was adopted. Phenolic acids were administrated orally in the initial stage of the model at a dose of 1 g·kg⁻¹ BW, which was combined withtherectal administration with three doses of supercritical extracts (15, 30, 60 g·kg⁻¹ BW). PCNA, 8-oxoguaine, γ-H2AX, iNOS and COX-2 were tested by immunohistochemistry and Western blot assays. The results showed that the combined administration of phenolic acids and supercritical extracts from A. sinensis suppressed the tumor growth and cell proliferation, and DNA damages and inflammatory responses were reduced in a dose-dependent manner. These results indicate that the combined administration of phenolic acids and supercritical extracts from A. sinensis have a certain effect in preventing carcinogenesis.

Towards a modern approach to traditional use of Helichrysum italicum in dermatological conditions: In vivo testing supercritical extract on artificially irritated skin.

ETHNOPHARMACOLOGICAL RELEVANCE: Helichrysum italicum has been widely used in traditional medicine to treat allergies, colds, cough, skin, liver and gallbladder disorders, inflammation, infections, and sleeplessness. Furthermore, it possesses considerable wound healing and skin protective properties, documented by several in vivo studies performed on animals. However, there is a lack of experimental evidence supporting its potential as a topical agent tested by human clinical trials. AIM OF THE STUDY: The study aimed to investigate the skin protective activity of cotton gauze and polypropylene non-woven fabric, impregnated with H. italicum extract by the integrated supercritical CO2 extraction-supercritical solvent impregnation process. MATERIALS AND METHODS: The integrated process of supercritical CO2 extraction of H. italicum and the impregnation of cotton gauze and polypropylene non-woven fabric was performed under 350 bar and 40 °C with and without the addition of ethanol as a cosolvent. Impregnated textile materials were tested in vivo for their bioactivity on irritated human skin. Randomized in vivo studies performed involved assays of both safety and efficacy of the impregnated textiles. The effects were evaluated using the in vivo non-invasive biophysical measurements of the following skin parameters: electrical capacitance, transepidermal water loss, melanin index, erythema index, and skin pH. RESULTS: Both cotton gauze and polypropylene non-woven fabric were impregnated with H. italicum extracts under supercritical conditions with considerable values of the impregnation yield (1.97%-4.25%). The addition of ethanol as a cosolvent during the process caused significant changes in the incorporated extracts' impregnation yield and chemical profile. Both impregnated textile materials were safe, evaluated by their testing on the human skin with no cause of any irritation and redness. However, efficacy studies revealed that polypropylene non-woven fabric impregnated with H. italicum extract with ethanol as a cosolvent, possessed significantly greater potential for skin protection than the other investigated samples. CONCLUSIONS: The present study demonstrated the feasibility of the combined supercritical extraction and impregnation process in developing materials for topical application based on H. italicum extract. The results of in vivo studies performed on human volunteers confirmed the suitability of H. italicum active components to be a part of human skin protective preparations because of their ability to maintain the skin unimpaired. Traditionally claimed applications as a medicinal plant capable of regenerating skin have been scientifically proven, in addition to employing green technology in obtaining the impregnated materials with a broad spectrum of utilization.

Effect of Supercritical Extract from Black Poplar and Basket Willow on the Quality of Natural and Probiotic Drinkable Yogurt.

Yogurt is a fermented milk drink produced by Streptococcus thermophilus, Lactobacillus delbrüeckii ssp. bulgaricus, or Lactobacillus rhamnosus, which can be enriched with polyphenolic compounds to enhance its antioxidant properties. Supercritical (scCO2/H2O) extracts obtained from the mixture of bark and wood of black poplar (Populus nigra) and basket willow (Salix viminalis) are the source of bioactive compounds. The aim of the study was to assess the effect of supercritical extracts from the P. nigra and S. viminalis on the fermentation, quality, and bioactive properties of drinkable natural and probiotic yogurts. The incorporation of scCO2/H2O extracts at a dose of 0.01% (w/v) into milk for the production of natural and probiotic yogurts increases their functional properties by enhancing the antioxidant activity without causing negative effects on the physicochemical and organoleptic properties of products. The antioxidant activity of yogurt with scCO2/H2O extract from P. nigra and S. viminalis was higher than control yogurts by 1.3-13.2% and 4.4-37.5%, respectively. The addition of a supercritical S. viminalis extract reduced the time of natural and probiotic yogurt fermentation. Natural and probiotic yogurt with scCO2/H2O extracts added was characterised by a bacterial population size of over 7 log cfu/g, and the microflora was active throughout the cold storage period. FTIR analysis confirmed the presence of scCO2/H2O extracts from P. nigra or S. viminalis in both types of yogurt. A secondary structure analysis confirmed interactions between compounds of scCO2/H2O extract from P. nigra and S. viminalis extract with milk proteins. These interactions affect the compounds' structural and functional properties by changing, e.g., their digestibility and antioxidant properties.

In-vitro and in silico activity of Cyamopsis tetragonoloba (Gaur) L. supercritical extract against the dengue-2 virus.

Our health and wealth are highly influenced by a number of viruses. Dengue is one of them having a global influence in absence of vaccines and antiviral. WHO suggested that the morbidity of dengue is increasing more than 6 times from 0.5 million in 2010 to over 3.34 million in 2016, following a sharp increase in 2019. The aim of the present study is to check the in vitro and in silico anti-dengue activity of Cyamopsis tetragonoloba supercritical extract in cell lines. The optimum yield of supercritical extract was obtained 0.13 g/10 g (1.3% w/w) at 40 °C temp and 15 MPa pressure and further characterized by GC-MS. The antiviral assay was performed on C6/36 cell lines with 100 copies of dengue-2 virus and maximum non-toxic dose (31.25 µg/ml) of supercritical extract and their effect was detected by real-time RT-PCR. This study revealed that C. tetragonoloba supercritical extract inhibited the dengue-2 virus (99.9%). GC-MS analysis of C. tetragonoloba supercritical extract showed the presence of 10 compounds. The major compounds identified were Hexadecanoic acid, 15-methyl-methyl ester (24.498%); 9,12-octadecadienoyl chloride, (z,z)- (23.718%); methyl dodecanoic acid (13.228%); methyl-stearate (8.696%); Tridecanoic acid, 12-methyl-, methyl-ester (8.426%), dodecanoic acid (6.102%). The study reveals that C. tetragonoloba can be exploited to develop an effective, inexpensive, and specific anti-dengue. The molecular docking study demonstrated the binding energy of 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (- 4.1 kcal/mol), 9,12-octadecadienoyl chloride (z,z) (- 4.0 kcal/mol) ligands were higher than others. It is concluded that C. tetragonoloba can play a major role to inhibit dengue-2 virus.

Tomato pomace extract and organic peppermint essential oil as effective sodium nitrite replacement in cooked pork sausages.

The effect of supercritical fluid extract of tomato pomace (TP) and essential oil of organic peppermint (PM) on pH, color, residual nitrite content, lipid oxidation (TBARS value) and total plate count (TPC) of cooked pork sausages produced with 50 mg/kg of sodium nitrite was investigated. Five batches were produced: T1: 100 mg/kg of sodium nitrite; T2: 50 mg of sodium nitrite; T3: 50 mg of sodium nitrite and 0.150 µL/g TP; T4: 50 mg of sodium nitrite, 0.075 µL/g TP and 0.075 µL/g PM; T5: 50 mg of sodium nitrite and 0.150 µL/g PM. The lowest residual nitrite content and TBARS value were observed in treatment T4. The inclusion of TP increased redness of cooked pork sausages. TPC was the lowest in treatment T5. The results of this study showed that the addition of TP and PM enhanced quality of cooked sausages produced with reduced level of sodium nitrite.

Marigold Supercritical Extract as Potential Co-adjuvant in Pancreatic Cancer: The Energetic Catastrophe Induced via BMP8B Ends Up With Autophagy-Induced Cell Death.

The recent development of powerful "omics" technologies (genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has opened new avenues in nutritional sciences toward precision nutrition, which is a genotype-directed nutrition that takes into account the differential responses to nutritional interventions based on gene variation (nutrigenetics) and the effect of nutrients on gene expression (nutrigenomics). Current evidence demonstrates that up to one third of the deaths caused by cancer could be prevented by acting on key risk factors, with diet being one of the most important risk factors due to its association with obesity. Additional factors such as composition of gut microbiome, the immune system, and the nutritional status will have an impact on the final outcome. Nutrient components and bioactive compounds from natural sources can have an impact on cancer progression or even the risk of cancer development by regulating gene expression and/or associated risk factors such as obesity and chronic inflammation. Nowadays, among the different methods to produce natural extracts, the green technology of supercritical fluid extraction (SFE) is quite popular, with a special interest on the use of supercritical CO2 for the extraction of compounds with low polarity. The success of nutritional interventions based on the use of nutraceuticals requires several steps: (i) in vitro and preclinical demonstration of their antitumoral effects; (ii) knowledge of their mechanism of action and molecular targets, which will allow for identification of the specific subgroups of patients who will benefit from them; (iii) the study of genetic variants associated with the differential responses; and (iv) innovative approaches of formulations to improve the in vivo bioavailability of the bioactive ingredients. Herein, we investigate the antitumoral properties and mechanism of action of a supercritical CO2 extract from Calendula officinalis, commonly known as marigold (marigold SFE) in the context of pancreatic cancer. Mechanistically, marigold SFE induces the expression of BMP8B, which leads to an energetic catastrophe ending up with autophagy-induced cell death (AICD). As metabolic reprogramming is a well-recognized hallmark of cancer, the direct impact of marigold SFE on pancreatic cancer cell metabolism encourages further research of its potential as a coadjuvant in pancreatic cancer therapy. Finally, we discuss innovative formulation approaches to augment the clinical therapeutic potential of marigold SFE in nutritional interventions.

Yarrow Supercritical Extract Ameliorates the Metabolic Stress in a Model of Obesity Induced by High-Fat Diet.

Nowadays, obesity and its associated metabolic disorders, including diabetes, metabolic syndrome, cardiovascular disease, or cancer, continue to be a health epidemic in westernized societies, and there is an increased necessity to explore anti-obesity therapies including pharmaceutical and nutraceutical compounds. Considerable attention has been placed on the identification of bioactive compounds from natural sources to manage the metabolic stress associated with obesity. In a previous work, we have demonstrated that a CO2 supercritical fluid extract from yarrow (Yarrow SFE), downregulates the expression of the lipogenic master regulator SREBF1 and its downstream molecular targets FASN and SCD in a tumoral context. Since obesity and diabetes are strongly considered high-risk factors for cancer development, herein, we aimed to investigate the potential therapeutic role of Yarrow SFE in the metabolic stress induced after a high-fat diet in mice. For this purpose, 32 C57BL/6 mice were distributed in four groups according to their diets: standard diet (SD); SD supplemented with Yarrow SFE (SD + Yarrow); high-fat diet (HFD); and HFD supplemented with Yarrow SFE (HFD + Yarrow). Fasting glycemia, insulin levels, homeostasis model assessment for insulin resistance (HOMA-IR), lipid profile, gene expression, and lipid content of liver and adipose tissues were analyzed after three months of treatment. Results indicate improved fasting glucose levels in plasma, enhanced insulin sensitivity, and diminished hypercholesterolemia in the HFD + Yarrow group compared to the HFD group. Mechanistically, Yarrow SFE protects liver from steatosis after the HFD challenge by augmenting the adipose tissue buffering capacity of the circulating plasma glucose.

In vitro uptake and immune functionality of digested Rosemary extract delivered through food grade vehicles.

The digestion, absorption, uptake and bioavailability of a rosemary supercritical fluid extract encapsulated in oil in water emulsion were studied. Two emulsions with opposite surface charge were prepared, containing 7% canola oil, and either 2% lactoferrin or whey protein isolate. When absorption and uptake of carnosic acid and carnosol were followed on Caco-2 cell monolayers, there were no differences with protein type. However, when co-cultures of HT-29 MTX were employed, the presence of mucus caused a higher retention of carnosic acid in the apical layer for lactoferrin emulsions. The immune activity of the bioavailable fractions collected from cell absorption experiments was tested ex vivo on murine splenocytes. Although transport through the intestinal barrier models was low, the bioavailable fractions showed a significant effect on splenocytes proliferation. These results demonstrated the potential of using rosemary supercritical extract through protein stabilized oil in water emulsions, as a food with immunomodulatory functionality.

Improved neuroprotective effects by combining Bacopa monnieri and Rosmarinus officinalis supercritical CO2 extracts.

Ethnobotanical evidence suggests that herbs such as brahmi (Bacopa monnieri) and rosemary (Rosmarinus officinalis) may possess antioxidant and neuroprotective properties. We compared the antioxidant and neuroprotective effects of supercritical extract of Bacopa monnieri and rosemary antioxidant extract obtained from Rosmarinus officinalis as well as their combination to examine the effects on human glial (U-87 MG) and embryonic mouse hypothalamus cells. Bacopa monnieri extract, rosemary antioxidant extract, and their combination (1:1) are not cytotoxic in both glial and embryonic mouse hypothalamus cell lines up to 200 µg/mL concentration. The combination of extracts of Bacopa monnieri + rosemary antioxidant has better antioxidant potential and antilipid peroxidation activity than either agent alone. Although the extract of Bacopa monnieri + rosemary antioxidant showed almost similar inhibition of phospho tau expression as Bacopa monnieri or rosemary antioxidant extract alone, the combination has better inhibitory effect on amyloid precursor protein synthesis and higher brain-derived neurotrophic factor production in hypothalamus cells than single agents. These results suggest that the extract of Bacopa monnieri + rosemary antioxidant is more neuroprotective than Bacopa monnieri or rosemary antioxidant extract.

Anti-inflammatory effects of Houttuynia cordata supercritical extract in carrageenan-air pouch inflammation model.

Anti-inflammatory effects of Houttuynia cordata supercritical extract (HSE) were investigated in rat carrageenan-air pouch model. Oral administration of HSE (50-200 mg/kg) suppressed carrageenan-induced exudation and albumin leakage, as well as inflammatory cell infiltration at a high dose (200 mg/kg). Intraperitoneal injection of dexamethasone (2 mg/kg) only decreased exudation and cell infiltration, while indomethacin (2 mg/kg, i.p.) reduced exudate volume and albumin content without influence on the cell number. HSE lowered tumor-necrosis factor-α (TNF-α) and nitric oxide (NO), as well as prostaglandin E(2) (PGE(2)). Dexamethasone only reduced TNF-α and NO, while indomethacin decreased PGE(2). The results indicate that HSE exhibits anti-inflammatory effects by inhibiting both TNF-α-NO and cyclooxygenase-2-PGE(2) pathways.

Prospecção de bioativos farmacológicos em algas marinhas Rhodophyta e Heterokontophyta e avaliação de citotoxicidade / Prospection of pharmaceutical bioactive compounds in marine algae Rhodophyta and Heterokontophyta and cytotoxicity evaluation

Muitas drogas terapêuticas produzidas pela indústria farmacêutica são estruturas químicas isoladas de organismos encontrados na natureza ou moléculas baseadas nelas. Podem ser incluídas nesse grupo drogas isoladas de organismos marinhos, como corais, esponjas e algas marinhas, conhecidos como produtores de grandes quantidades de metabólitos secundários. Com base neste fato o presente estudo teve como objetivo realizar a prospecção de moléculas bioativas com propósito farmacológico, em extratos de algas marinhas vermelhas (Rhodophyta) e pardas (Heterokontophyta) coletadas no litoral brasileiro. A prospecção foi realizada por meio de avaliação de seus potenciais antioxidante, antibacteriano, antifúngico, anticancerígeno, e antiparasitário contra organismos causadores de leishmaniose e esquistossomose. Para as avaliações foram empregadas os extratos supercríticos de 5 espécies diferentes, sendo 2 pardas: Dictyota dichotoma e D. menstrualis e três vermelhas: Chondria littoralis, Spyridia hypnoides e Plocamium brasiliense. Os extratos foram avaliados quanto aos seus potenciais bioativos e os resultados mais promissores foram selecionados para as etapas seguintes do fracionamento. Em uma avaliação geral os extratos apresentaram bons resultados e representam uma potencial fonte de bioativos. Os extratos das espécies de D. dichotoma e D. menstrualis foram então submetidos a um procedimento de fracionamento bioguiado pela atividade esquistossomicida. Incorporou-se ainda um terceiro extrato de D. mertensii aos estudos e todas as etapas do fracionamento foram monitoradas por LC-MS. Comparando-se as massas detectadas em todas as frações que apresentaram atividade, para os 3 extratos, foi verificado que a substância de m/z 271,24 estava presente em todas elas, portanto os procedimentos de isolamento foram direcionados a esta molécula para a qual foi possível isolar 7 mg. Diferentemente do que era esperado a molécula quando avaliada isoladamente não apresentou atividade esquistossomicida, levando a hipótese de que a atividade seja decorrente de uma molécula diferente para cada espécie ou ainda que a mesma seja decorrente de uma interação com outras substâncias por um mecanismo de ação aditivo ou sinérgico. O trabalho avaliado de forma geral apresentou resultados promissores e representa um grande embasamento para servir como base para posteriores trabalho de fracionamento

Several therapeutic drugs manufactured by the pharmaceutical industry are chemical structures isolated from organisms that are found in nature or molecules based on that. May be included at this group drugs isolated from marine organisms, like corals, sponges and seaweeds, known as great secondary metabolites producers. Based on this facts the objective of the present study is to perform a prospection study to achieve bioactive molecules with pharmaceutical purposes, on extracts made from red (Rhodophyta) and brown (Heterokontophyta) seaweed collected in the Brazilian shore. The prospection studies was performed by means of evaluation of the antioxidant, antibacterial, antifungal, anticancer and antiparasitic (against Leishmania and Schistosoma) potential. In the evaluation were tested the supercritical extracts of 5 different species, including 2 brown species: Dictyota dichotoma and D. menstrualis and 3 red species: Chondria littoralis, Spyridia hypnoides and Plocamium brasiliense. The extracts were evaluated by their potential bioactive compounds and the most promising results were selected for the following fractionation steps. Overall the extracts have shown good results and may be represent a potential source of bioactive molecules. The extracts of both D. dichotoma and D. menstrualis were submitted to a bioguided fractionation process by their antischistosomal activities. It was still included a third extract from D. mertensii to the studies and every step was monitored by LC-MS techniques. Comparing the detected mass for each active fraction, it was observed the presence of a substance with m/z 271,24 in all of the extracts, so the isolating procedures were directed to obtain that specific molecule, which was obtained in a biomass of 7 mg. Differently than expected the molecule when evaluated isolated do not show the antischistosomal activity, leading to the hypothesis that the activity was related to different molecules for each species or even the observed effect is resulted by an interaction mechanism with another substances by an additive or synergist mechanism. The overall evaluation of the whole work show some promising results and it represent a great support for future fractionation works