Aqueous two-phase extraction of phenolic compounds from Sedum dendroideum with antioxidant activity and anti-proliferative properties against breast cancer cells

Highlights

• Ethanol-salt ATPS are suitable for recovering phenolic compounds from S. dendroideum.

• Volume ratio, sample load, NaCl addition and tie-line length affect the partition.

• The main driving force of phenolic compounds partition is the polarity of molecules.

• Phenolic compounds obtained by ATPS extraction show antioxidant activity.

• S. dendroideum extracts show anti-proliferative activity on breast cancer cells.

Abstract

Sedum dendroideum, a traditional medicinal plant, has a wide variety of pharmacological activities, attributed to phenolic compounds. Despite that there are several methods available for the extraction of phenolic compounds, their use has several disadvantages, evidencing the need for the development of alternative methods. In this study, ethanol-salt aqueous two-phase systems (ATPS) were employed for the extraction of phenolic compounds from S. dendroideum. The effect of different system parameters on the partition of phenolic compounds were studied, including the volume ratio (V_R), tie-line length (TLL), sample loading, and sodium chloride addition. Also, thermodynamics of partition process was addressed. Furthermore, systems top phase antioxidant activity and anti-proliferative properties against MCF-7 breast cancer cells were evaluated. A system with V_R 3, TLL 40% w/w, 5 mg of crude extract/g of ATPS, and 3% w/w NaCl, showed the highest phenolic compounds recovery in the top phase (17.98%). Thermodynamic studies indicated that polarity governs the partition of phenolic compounds. The system with a V_R 3, TLL 40% w/w, 5 mg of crude extract/g of ATPS, and without NaCl exhibited the highest antioxidant activity. HPLC analysis revealed no alteration on phenolic compounds profile after ATPS extraction. Whereas, the top phase of a system with V_R 3, TLL 40% w/w, 5 mg of crude extract/g of ATPS, and 5% w/w of NaCl showed maximum inhibition of cell proliferation at 168 h (89.66%). The potential application of S. dendroideum as an anticancer agent is reported for the first time. ATPS could be used as an alternative extraction technique for further evaluation of phytochemical composition and pharmacological activities of phenolic compounds from S. dendroideum.

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 (V_R) 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, V_R, 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.