Recovery of Polyphenols from Rosehip Seed Waste Using Natural Deep Eutectic Solvents and Ultrasonic Waves Simultaneously.

Rosehips are processed and consumed in numerous forms, such as juice, wine, herbal tea, yogurt, preserved fruit, and canned products. The seeds share in fruit is 30-35% and they have recently been recognized as an important source of oil rich in unsaturated fatty acids. However, after defatting, seed waste may still contain some polar polyphenolic compounds, which have been scarcely investigated. The aim of this study was to examine the potential of the defatted seed waste as a source of polyphenols. For the defatting process, supercritical carbon dioxide extraction at 300 bar and 40 °C was applied. The capacity of eight different natural deep eutectic solvents (NADES) for the recovery of phenolics from defatted rosehip seed powder (dRSP) was examined. In the extracts obtained with ultrasound-assisted NADES extraction, twenty-one phenolic compounds were identified with LC-MS/MS, among which the most abundant were quinic acid (22.43 × 103 µg/g dRSP) and catechin (571.93 µg/g dRSP). Ternary NADES formulations based on lactic acid proved to be superior. Potential correlations between identified chemical compounds, solvent polarity and viscosity, as well as the compound distributions across studied solvent combinations in PCA hyperspace, were also investigated. PCA demonstrated that more polar NADES mixtures showed improved extraction potential. The established environmentally friendly process represents an approach of transforming rosehip seed waste into value-added products with the potential to be applied in the food industry and to contribute to sustainable production.

A new green approach for Lavandula stoechas aroma recovery and stabilization coupling supercritical CO2 and natural deep eutectic solvents.

This work investigated a green approach to obtain and stabilize Lavandula stoechas L. volatile organic compounds with sensory aroma characteristics by using alternative solvents, namely supercritical carbon dioxide (scCO2) and deep eutectic solvents (DES). The CO2 extracts were dispersed in different DES mixtures (betaine:ethylene glycol (1:3), betaine:glycerol (1:2), and glycerol:glucose (4:1)) and their stability was monitored during 6 months of storage at room temperature by monitoring the headspace (HS) profile. The CO2 extract was used as the control. It was initially determined that there was a dominant presence of oxygenated monoterpenes (67.33-77.50%) in the extracts. During storage, significant changes occurred in the samples' HS, such as the decrease in terpene hydrocarbons which also affected the presence of oxygenated terpenes, which increased in certain cases. Moreover, the highest formation of new components was recorded in the control which could be an indicator of decreased stability. The DESs-CO2 were more stable than the CO2 control and among them, betaine:ethylene glycol stood out as the most adequate systems for maintaining the stability of L. stoechas HS components. For the visual estimation of similarities and dissimilarities among the samples, chemometric pattern recognition approaches were applied including the hierarchical cluster analysis, principal component analysis, and sum of ranking differences.

Innovative Strategy for Aroma Stabilization Using Green Solvents: Supercritical CO2 Extracts of Satureja montana Dispersed in Deep Eutectic Solvents.

The aim of this work was to establish the potential of natural deep eutectic solvents (NADES) for the stabilization of aroma volatile organic compounds from a natural source. Satureja montana was used as a source of volatile components, as it is rich in terpenes of great commercial and biological importance, such as carvacrol, thymol, and thymoquinone, among others. Supercritical CO2 was used to extract the lipophilic fraction of S. montana, which was further directly dispersed in NADES. The stabilizing capacity of seven different NADES based on betaine and glycerol was analyzed. The stability of the components in NADES was monitored by analyzing the headspace profile during 6 months of storage at room temperature. The changes in the headspace profile over time were analyzed by using different statistical and chemometric tools and the Wilcoxon matched pair test. It was determined that alterations over time occurred such as degradation and oxidation, and they were the most prominent in the control. In addition, the indicator of decreased stability of the control was the formation of the new compounds that could compromise the quality of the product. In the stabilized NADES samples, the changes were significantly less prominent, indicating that the NADES had a stabilizing effect on the volatile compounds. According to Wilcoxon matched pair test, the most efficient stability was achieved by using betaine/ethylene glycol, glycerol/glucose, and betaine/sorbitol/water. Therefore, by applying two green solvents, a sustainable approach for obtaining pure and high-quality S. montana extracts with extended stability at room temperature was established.

Towards a Greener Approach for Biomass Valorization: Integration of Supercritical Fluid and Deep Eutectic Solvents.

A green and sustainable procedure for obtaining Lavandula stoechas extracts with antioxidant and antimicrobial properties was investigated. Green solvents, supercritical CO2, and natural deep eutectic solvents (NADES) together with ultrasound-assisted extraction were used for the sequential extraction of terpene and polyphenols fractions. After the CO2 extraction of the terpene fraction, the residue material was used in an extraction with different NADES (betaine-ethylene glycol (Bet:EG), betaine-glycerol (Bet:Gly), and glycerol-glucose (Gly:Glu)), intensified with an ultrasound-assisted method (at 30 and 60 °C). In the CO2 extract, the major group of components belonged to oxygenated monoterpenes, while the highest polyphenol content with the dominant rutin (438.93 ± 4.60 µg/mL) was determined in Bet:EG extracts (60 °C). Bet:EG extracts also exhibited the most potent antioxidant activity according to DPPH, ABTS, and FRAP assays. Moreover, Bet:EG extracts showed significant inhibitory activity against Gram-positive and Gram-negative bacteria, with minimum inhibitory activity of 0.781-3.125 and 1.563-6.250 mg·mL-1, respectively. By comparing the polyphenolic content and antioxidant and antimicrobial activities of Bet:EG extracts with extracts obtained with conventional solvents (water and ethanol), the superiority of NADES was determined. The established environmentally friendly procedure unifies the requirements of green and sustainable development and modern pharmacognosy because it combines the use of safe alternative solvents, the absence of solvent waste generation, more rational use of resources, and the attainment of safe and quality extracts.

Application of Green Technology to Extract Clean and Safe Bioactive Compounds from Tetradesmus obliquus Biomass Grown in Poultry Wastewater.

Microalgae are capable of assimilating nutrients from wastewater (WW), producing clean water and biomass rich in bioactive compounds that need to be recovered from inside the microalgal cell. This work investigated subcritical water (SW) extraction to collect high-value compounds from the microalga Tetradesmus obliquus after treating poultry WW. The treatment efficiency was evaluated in terms of total Kjeldahl nitrogen (TKN), phosphate, chemical oxygen demand (COD) and metals. T. obliquus was able to remove 77% TKN, 50% phosphate, 84% COD, and metals (48-89%) within legislation values. SW extraction was performed at 170 °C and 30 bar for 10 min. SW allowed the extraction of total phenols (1.073 mg GAE/mL extract) and total flavonoids (0.111 mg CAT/mL extract) with high antioxidant activity (IC50 value, 7.18 µg/mL). The microalga was shown to be a source of organic compounds of commercial value (e.g., squalene). Finally, the SW conditions allowed the removal of pathogens and metals in the extracts and residues to values in accordance with legislation, assuring their safety for feed or agriculture applications.

Evaluation of In Vitro Cytotoxic Potential of Avarol towards Human Cancer Cell Lines and In Vivo Antitumor Activity in Solid Tumor Models.

The goal of this study was to determine the activity in vitro and in vivo of avarol, a sesquiterpene hydroquinone originating from the Dysidea avara sponge from the south Adriatic Sea, against different cancer cell lines and two types of mouse carcinoma. To investigate the in vitro cytotoxicity, a human cervix adenocarcinoma cell line (HeLa), human colon adenocarcinoma (LS174), human non-small-cell lung carcinoma (A549), and a normal human fetal lung fibroblast cell line (MRC-5) were used. The in vivo antitumor activity was investigated against two transplantable mouse tumors, the Ehrlich carcinoma (EC) and cervical cancer (CC-5). The effect of avarol on cancer cell survival, which was determined by the microculture tetrazolium test, confirmed a significant in vitro potency of avarol against the investigated cell lines, without selectivity towards MRC-5. The highest cytotoxicity was exhibited against HeLa cancer cells (10.22 ± 0.28 µg/mL). Moreover, potent antitumor activity against two tumor models was determined, as the intraperitoneal administration of avarol at a dose of 50 mg/kg resulted in a significant inhibition of tumor growth in mice. After three administrations of avarol, a 29% inhibition of the EC growth was achieved, while in the case of CC-5, a 36% inhibition of the tumor growth was achieved after the second administration of avarol. Therefore, the results indicate that this marine sesquiterpenoid hydroquinone could be a promising bioactive compound in the development of new anticancer medicine.

The Application of Ultrasonic Waves and Microwaves to Improve Antihyperglycaemic and Antimicrobial Activities of Marrubium vulgare Extracts.

Having scarce information about ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE) of white horehound (Marrubium vulgare L.), the idea has emerged to determine the optimal process parameters for the maximization of polyphenols and to compare the efficiency of these green extraction technologies. The optimal UAE parameters are temperature of 73.6 °C, extraction time of 40 min and ultrasound power of 30.3 W/L, while the optimal MAE parameters are 63.8% ethanol, extraction time of 15 min and microwave power of 422 W. Extract obtained at optimal UAE parameters shows the highest antihyperglycemic activity (α-amylase inhibition: 50.63% and α-glucosidase inhibition: 48.67%), which can potentially be explained by the presence of chlorogenic acid and quercetin, which were not identified in the macerates. The most sensitive bacterial strain to optimal ultrasonic extract is Bacillus cereus, whereas the most sensitive fungal strain is Saccharomyces cerevisiae.

The Effects of Resveratrol-Rich Extracts of Vitis vinifera Pruning Waste on HeLa, MCF-7 and MRC-5 Cells: Apoptosis, Autophagia and Necrosis Interplay.

Resveratrol is a well-studied plant-derived molecule in cancer biology, with a plethora of documented in vitro effects. However, its low bioavailability and toxicity risk hamper its wider use. In this study, vine shoots after pruning were used as a source of resveratrol (RSV). The activity of subcritical water extract (SWE) and dry extract (DE) is examined on three cell lines: HeLa, MCF-7 and MRC-5. The cytotoxic effect is assessed by the MTT test and EB/AO staining, levels of apoptosis are determined by Annexin V assay, autophagia by ULK-1 expression using Western blot and NF-kB activation by p65 ELISA. Our results show that both resveratrol-rich extracts (DE, SWE) have a preferential cytotoxic effect on malignant cell lines (HeLa, MCF-7), and low cytotoxicity on non-malignant cells in culture (MRC-5). Further experiments indicate that the investigated malignant cells undergo different cell death pathways. MCF-7 cells died preferentially by apoptosis, while the HeLa cells died most likely by necrosis (possibly ferroptosis). Protective autophagia is diminished upon treatment with DE in both HeLa and MCF-7 cells, while SWE does not influence the level of autophagia. The extracts are effective even at low concentrations (below IC50) in the activation of NF-kB (p65 translocation).

Supercritical CO2 Extract from Microalga Tetradesmus obliquus: The Effect of High-Pressure Pre-Treatment.

High-pressure pre-treatment followed by supercritical carbon dioxide (ScCO2) extraction (300 bar, 40 °C) was applied for the attainment of the lipophilic fraction of microalga Tetradesmus obliquus. The chemical profile of supercritical extracts of T. obliquus was analyzed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). Moreover, the impact of ScCO2 on the microbiological and metal profile of the biomass was monitored. The application of the pre-treatment increased the extraction yield approximately three-fold compared to the control. In the obtained extracts (control and pre-treated extracts), the identified components belonged to triacylglyceroles, fatty acid derivatives, diacylglycerophosphocholines and diacylglycerophosphoserines, pigments, terpenes, and steroids. Triacylglycerols (65%) were the most dominant group of compounds in the control extract. The pre-treatment decreased the percentage of triacylglycerols to 2%, while the abundance of fatty acid derivatives was significantly increased (82%). In addition, the pre-treatment led to an increase in the percentages of carotenoids, terpenoids, and steroids. Furthermore, it was determined that ScCO2 extraction reduced the number of microorganisms in the biomass. Considering its microbiological and metal profiles, the biomass after ScCO2 can potentially be used as a safe and important source of organic compounds.

Comparative Study of the Essential Oil and Hydrosol Composition of Sweet Wormwood (Artemisia annua L.) from Serbia.

The most abundant volatile compounds of sweet wormwood (Artemisia annua L.) essential oil were artemisia ketone (25.4 %) and trans-caryophyllene (10.2 %), followed by 1,8-cineole, camphor, germacrene D and ß-selinene. The major volatile compounds in the hydrosol were camphor (25.1 %), 1,8-cineole (20.5 %) and artemisia ketone (10.7 %), followed by trans-pinocarveol and yomogi alcohol. Tested essential oil was rich in oxygenated monoterpenes and sesquiterpene hydrocarbons, while the former were identified as the major class of volatile compounds in the hydrosol, due to higher water solubility. Classification of all sweet wormwood chemotypes, according to essential oil composition, in available literature (17 studies and 61 accessions) could be done according to four chemotypes: artemisia ketone+artemisia alcohol (most abundant), artemisia ketone, camphor and nonspecific chemotype. According to this classification, essential oil of sweet wormwood from this study belongs to artemisia ketone (content varied between 22.1 and 55.8 %). Bearing in mind that hydrosols are a by-product of industrial production of essential oils, and the fact that sweet wormwood hydrosol has high contents of camphor, 1,8-cineole and artemisia ketone, there is a great potential for the use of this aromatic plant primary processing waste product as a water replacement in cosmetic industry, beverages flavoring, for food preservation, as well as in post-harvest pre-storage treatments in organic agriculture.

Effect of Type and Concentration of Carrier Material on the Encapsulation of Pomegranate Peel Using Spray Drying Method.

This study aimed to establish a procedure for pomegranate peel (PP) valorization and attainment of stable extracts with preserved bioactive compounds. The technology applied was spray drying with carbohydrate-based (maltodextrin, MD) and protein-based (whey protein, WP) carrier materials in different concentrations (80, 100, and 120%). What was analyzed was the impact of the type and concentration of carrier material on the stability and quality of the final encapsulated powder. The best results were achieved when the PP extract was microencapsulated with the carbohydrate-based carrier (100%), where it had the highest encapsulation efficiency (EE) (88.63%), hygroscopicity (15.17%), and water solubility index (87.04%). The moisture content was in the range of 3.69-4.60% and 4.21-5.84% for MD and WP, respectively, indicating that both are suitable for long-term storage. It was observed that changes in carrier concentration significantly influenced most of the powders' physicochemical properties. Microencapsulation using MD yielded a higher content of punicalin, punicalagin, gallic, and ellagic acid than those with WP. Overall results demonstrated that carbohydrate-based microencapsulation can be utilized efficiently for the protection of powder stability and phytochemical characteristics.

Optimization of MAE for the Separation of Nicotine and Phenolics from Tobacco Waste by Using the Response Surface Methodology Approach.

This study intends to valorize by-products of the industrial processing of tobacco to obtain nicotine and phenolics as value-added compounds. Three influential parameters of the microwave-assisted extraction-MAE (temperature, treatment time, and solvent/solid ratio) were studied for the optimization of the extraction protocol for tobacco leaves and three types of waste-scrap, dust, and midrib, respectively. Nicotine was the dominant bioactive compound in all extracts, ranging from 1.512 to 5.480% in leaves, 1.886 to 3.709% in scrap, 2.628 to 4.840% dust, and 0.867 to 1.783% in midrib extracts. Five phenolic compounds were identified and quantified, predominated by chlorogenic acid and rutin. Additionally, total phenol content and antioxidant activity were determined using spectrophotometric assays. Optimization was performed in two aspects: to obtain a maximum extraction yield with minimum nicotine content and to obtain a maximum extraction yield with maximum nicotine content. These findings demonstrate that tobacco waste is a valuable source of bioactive compounds and MAE can be a promising alternative technique to obtain extracts rich in targeted bioactive compounds, especially nicotine.

Supercritical Carbon Dioxide Extraction of Allium ursinum: Impact of Temperature and Pressure on the Extracts Chemical Profile.

The aim of this study was to extract Allium ursinum L. for the first time by supercritical carbon dioxide (SC-CO2 ) as green sustainable method. The impact of temperature in the range from 40 to 60 °C and pressure between 150 and 400 bar on the quality of the obtained extracts and efficiency of the extraction was investigated. The highest extraction yield (3.43 %) was achieved by applying the extraction conditions of 400 bar and 60 °C. The analysis of the extracts was performed by gas chromatography and mass spectrometry (GC/MS). The most dominant sulfur-containing constituent of the extracts was allyl methyl trisulfide with the highest abundance at 350 bar and 50 °C. In addition, the presence of other pharmacologically potent sulfur compounds was recorded including S-methyl methanethiosulfinate, diallyl trisulfide, S-methyl methylthiosulfonate, and dimethyl trisulfide. Multivariate data analysis tool was utilized to investigate distributions of the identified compounds among the extracts obtained under various extraction conditions and yields. It was determined that the SC-CO2 extraction can by efficiently used for A. ursinum.

Supercritical CO2 extraction of Marrubium vulgare: intensification of marrubiin.

Supercritical carbon dioxide and Soxhlet extraction were employed for delivering Marrubium vulgare extracts. By varying process temperature and pressure, the impact of different densities of carbon dioxide on the extraction yield and chemical profile of the extracts was investigated. The highest extraction yields of M. vulgare were obtained by supercritical carbon dioxide extraction (3.51%) at 300 bar and 60 °C, while the yield obtained with the Soxhlet extraction was 3.23%. The chemical profiles of these two extracts were significantly different and marrubiin, the most dominant component of supercritical extracts was not present in extracts obtained by this conventional method. This labdane diterpene was the most abundant in extracts obtained at 200 bar and temperatures of 40, 50, and 60 °C (62.60, 68.41, and 71.96%, respectively). For the intensification of marrubiin, supercritical fractions were collected in successive time intervals over the course of the extraction (300 bar/60 °C). It was determined that after 1 h of extraction the highest content of marrubiin (75.14%) can be achieved. The similarities between the obtained extracts were estimated and the correlations to the content of identified lipophilic compounds were established using multivariate data analysis tools.

Enzymatic and Microwave Pretreatments and Supercritical CO2 Extraction for Improving Extraction Efficiency and Quality of Origanum vulgare L. spp. hirtum Extracts.

The goal of the study was to establish a procedure for improving the efficiency of supercritical carbon dioxide (scCO2) extraction of Origanum vulgare L. spp. hirtum (Greek oregano) and enhancing the quality of obtained extracts. Microwave and enzymatic pretreatments of the plant material were applied prior to the scCO2 extraction. It was determined that the microwave pretreatment with irradiation power 360 W during 2 min accelerated the extraction of lipophilic compounds and provided a twofold higher extraction yield compared to the control. Moreover, this pretreatment also led to an increase in oxygenated monoterpenes content and the most dominant component carvacrol, as well as the extracts' antioxidant activity. The enzymatic pretreatment caused a significant increase in the extraction yield and the attainment of the extract with the most potent antioxidant properties. Coupling the pretreatments with scCO2 extraction improves the process of obtaining high value lipophilic products of oregano in terms of utilization of the plant material, acceleration of the extraction with the possibility to adjust its selectivity and quality of extracts, and enhancement of biological activity.

Evaluation of Anticancer Activity of Satureja montana Supercritical and Spray-Dried Extracts on Ehrlich's Ascites Carcinoma Bearing Mice.

Satureja montana herbal species belongs to aromatic medicinal plants with a significant place in traditional medicine. However, products produced with conventional procedures do not meet the requirements of the modern market which include environmentally-safe processes that provide quality, safe, and standardized products. In this study, the antiproliferative activity of S. montana extracts obtained by supercritical carbon dioxide and solid-liquid extraction followed by spray drying was investigated using the in vivo model of Ehrlich ascites carcinoma (EAC) in mice. The impact of two concentrations of extracts on the growth of tumor and the redox status of malignant cells was monitored. It was determined that the extracts induced oxidative stress in the malignant cells which was confirmed by the changes in activity of biochemical indicators of oxidative stress. The posttreatment was not an efficient approach, while the extracts applied as pretreatment and treatment resulted in an increase in the xanthine oxidase (XOD) activity, a decrease in catalase (CAT) activity, and an increase in the intensity of lipid peroxidation (LPx). Furthermore, a decrease in the values of reduced glutathione (GSH) and an increase in glutathione reductase (GR) and glutathione peroxidase (GSHPx) in EAC cells were recorded.

Comparative Study of Subcritical Water and Microwave-Assisted Extraction Techniques Impact on the Phenolic Compounds and 5-Hydroxymethylfurfural Content in Pomegranate Peel.

Two environmentally friendly innovative extraction techniques - subcritical water (SWE) and microwave-assisted extraction (MAE) were applied for the extraction of phenolics from pomegranate peel. The impact of process conditions (SWE: temperature 100-220 °C, extraction time 5-30 min; MAE: solvent water and 50% ethanol, irradiation power 470 and 800 W) on the quality of extracts in terms of the content of total phenolics, total flavonoids, major phenolic constituents (gallic acid, ellagic acid, punicalin, punicalagin), as well as 5-hydroxymethylfurfural(HMF) amount was investigated. For SWE, temperature of 130 °C and 20 min extraction time were found optimal for obtaining high content of bioactive compounds and minimizing the yield of HMF. During MAE, phenolic compounds were effectively extracted by using lower microwave power and 50% ethanol. Comparing two techniques, MAE is more efficient than SWE for the extraction of phenolics from pomegranate peel while obtaining a HMF-free extracts.

Valorization of Yarrow (Achillea millefolium L.) By-Product through Application of Subcritical Water Extraction.

In the present study, valorization of yarrow (Achillea millefolium) by-product from the filter tea industry was investigated through the application of subcritical water for the extraction of bioactive compounds. The influence of different process parameters (temperature 120-200 °C, extraction time 10-30 min, and HCl concentration in extraction solvent 0-1.5%) on extract quality in terms of content of bioactive compounds and antioxidant activity was investigated. Optimal conditions of the extraction process (temperature 198 °C, extraction time 16.5 min, and without acidifer) were determined and, when applied, the most efficient exploitation of by-products is achieved, that is, attainment of extracts rich in total phenols and flavonoids and high antioxidant activity. In addition, by applying the high performance liquid chromatographic analysis, the content of chlorogenic acid was determined as well as the hydroxymethylfurfural content in obtained extracts. The results demonstrated that subcritical water can be successfully used for utilization of yarrow by-products for obtaining extracts rich in antioxidants.

Application of Deep Eutectic Solvents for the Extraction of Rutin and Rosmarinic Acid from Satureja montana L. and Evaluation of the Extracts Antiradical Activity.

Satureja montana L. was used in the current research as the plant exhibits numerous health-promoting benefits due to its specific chemical composition. The extraction method based on deep eutectic solvents (DESs) was used for the extraction of rutin and rosmarinic acid from this plant. Five different choline chloride-based DESs with different volumes of water (10%, 30%, and 50% (v/v)) were used for the extraction at different temperatures (30, 50, and 70 °C) to investigate the influence on rosmarinic acid and rutin content obtained by high-performance liquid chromatography with diode-array detector (HPLC-DAD) in the obtained extracts. A principal component analysis was employed to explore and visualize the influence of applied parameters on the efficiency of the extraction procedure of rutin and rosmarinic acid. Among the tested DESs, choline chloride:lactic acid (mole ratio 1:2) and choline chloride:levulinic acid (mole ratio 1:2) were the most suitable for the extraction of rutin, while for rosmarinic acid choline chloride:urea (mole ratio 1:2) was the most effective solvent. The extract showing the best antiradical activity was obtained with choline chloride:urea (mole ratio 1:1) at 30 °C and 50% H2O (v/v).