Aqueous two-phase extraction of phenolic compounds from Sedum dendroideum with antioxidant activity and anti-proliferative properties against breast cancer cells
Introduction
Medicinal plants are widely recognized as a source of bioactive compounds for drug production due to their chemical diversity [1]. Sedum is a genus that comprises about 100 plant species from the Crassulaceae family; many of them have been used for medicinal purposes [2]. Sedum dendroideum, known as “Siempreviva”, is a small shrub with yellow flowers found from Mexico to Guatemala and Brazil [3], [4]. In traditional medicine this species has several applications, among them the treatment of eye conditions such as pterygium, pain, and inflammation [4], [5]. It is also referred as a remedy in mouth diseases, typhoid, dysentery, scurvy, headache, fever, venereal diseases or as a contraceptive [5]. Previous studies have shown several pharmacological activities of S. dendroideum extracts including antinociceptive, antioxidant, antiulcer, anti-inflammatory, spermicidal, and antidiabetic [3], [4], [6], [7], [8], [9], [10]. Those medicinal properties have been mainly attributed to diverse phenolic compounds including phenols, tannins, and flavonoids [3], [8], [9], [10].
The primary recovery of phenolic compounds from plants is commonly performed by liquid–liquid and solid–liquid extraction [11]. Multiple solvents are used for this purpose, however highly polar solvents, such as methanol and ethanol, have shown higher effectiveness [12]. To avoid problems associated with high solvent consumption, long processing times and significant energy consumption in conventional extraction techniques, more advanced techniques have been used, such as supercritical fluid extraction, microwave assisted extraction, ultrasonic assisted extraction and pressurized fluid extraction [13], [14], [15], [16]. However, the application of these novel techniques also involves higher costs, requirement of special equipment and rigorous operating conditions [17]. Alternatives such as aqueous two-phase systems (ATPS) have been proposed for the primary recovery of phenolic compounds [17], [18], [19], [20], [21], [22], [23].
ATPS is a liquid–liquid extraction method that has been used for the recovery of a wide variety of bioproducts, including proteins, cells, genetic material, virus, organelles, and low molecular weight compounds [24]. The use of ATPS has several advantages, such as easy scale up, short processing time, low cost, process integration capability, and higher recovery for target compounds in a single step [20], [25]. ATPS are formed by mixing aqueous solutions of two immiscible liquids at a certain critical concentration. The partition behavior of the molecules in the system depends on its design parameters such as tie-line length (TLL), volume ratio (VR) and sample loading; besides the selection of the system constituents. The physicochemical properties of the molecules also play a significant role in the partition behavior [24]. Particularly, alcohol-salt ATPS are of great interest for recovering phenolic compounds from plant tissue due to their high polarity. Additionally, this type of ATPS has a low toxicity, which seems to be adequate when the target is recovered for a medicinal application [26]. Several reports have shown the feasibility of using alcohol-salt systems for the extraction of different types of phenolic compounds from plants, such as anthocyanins from mulberry fruit, flavonoids from Ginkgo biloba leaves and pigeon pea roots, and chlorogenic acid from stressed carrot tissue roots [17], [20], [21], [27]. However, to our knowledge there is no information about the extraction of phenolic compounds from S. dendroideum using alcohol-salt ATPS.
In this study, the use of ethanol-salt ATPS for the recovery of phenolic compounds from S. dendroideum was investigated. The sample load, VR, TLL and neutral salt addition were varied to analyze the influence of these system parameters upon phenolic compounds recovery. Furthermore, a thermodynamic analysis was done to determine the main partition driving force of phenolic compounds. Taking into consideration the medicinal properties attributed to phenolic compounds in S. dendroideum, antioxidant activity of the compounds extracted by ATPS was evaluated, as well as anti-proliferative activity in a cancer cell line.
Section snippets
Chemicals and reagents
Ethanol (96%), potassium persulphate, phosphoric acid, dibasic potassium phosphate (K2HPO4) and monobasic potassium phosphate (KH2PO4) were obtained from D.E.Q. (Monterrey, NL, Mexico). 2,2́-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Folin-Ciocalteu reagent, methanol, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham, dimethyl sulfoxide (DMSO), sodium carbonate and sodium chloride
Effect of system parameters in phenolic compounds partition behavior and recovery
In order to characterize the partition behavior of phenolic compounds in ethanol-salt ATPS, different design parameters such as VR, TLL, sample loading and NaCl addition were evaluated. For further applications, the salt and ethanol presented along with the phenolic compounds had to be removed. Ethanol was easily removed from the top phase through evaporation. Whereas, salt removal from both phases was performed using dilution crystallization. This approach proved to be effective for the top
Conclusions
In this study, aqueous two-phase extraction, antioxidant and antiproliferative activity of phenolic compounds from S. dendroideum is reported. It was shown that system parameters, such as VR, TLL, sample load and NaCl addition, influenced the partition behavior of phenolic compounds. To increase the recovery of phenolic compounds in the top phase, using a smaller TLL, VR and sample load proved to be more adequate. Furthermore, the addition of NaCl allowed to increase phenolic compounds recovery
CRediT authorship contribution statement
Daniela Enriquez-Ochoa: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Visualization. Calef Sánchez-Trasviña: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. Betsabé Hernández-Sedas: Methodology, Investigation. Karla Mayolo-Deloisa: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition. Judith Zavala: Methodology, Resources,
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgements
The authors thank the Translational Omics Focus Group and the financial support provided by the Bioengineering and Regenerative Medicine (Grant 0020609M07) and Innovative Therapies in Visual Sciences Focus Groups (Grant 0020209M05) from Tecnologico de Monterrey. Daniela Enriquez-Ochoa and Calef Sánchez-Trasviña acknowledge CONACyT for the respectively awarded scholarships 712327 and 492276. Special thanks are due to Erika Ortega-Hernández from Tecnologico de Monterrey for her valuable technical
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