Extraction of Morus alba leaves using supercritical CO₂ and ultrasound-assisted solvent: Evaluation of β-sitosterol content

Highlights

• Supercritical CO₂ and ultrasound-assisted extraction of Morus alba leaves.

• The increase of pressure improved the yield of supercritical extraction.

• Ultrasound-assisted extraction was performed using a 2³ experimental design.

• β-Sitosterol was one of major constituents of the M. alba leaves extract.

• Supercritical CO₂ extraction provided up to 155.74 mg β-sitosterol 100 g⁻¹ leaves.

Abstract

In this study, extracts of Morus alba leaves were obtained by supercritical CO₂ and ultrasound-assisted methods, and the results were compared to those of the conventional Soxhlet extraction. The supercritical extraction resulted in the highest yields of 1.08 and 1.11 wt%, when under the highest pressure conditions investigated (200 bar; 40 and 60 °C, respectively). The ultrasound-assisted n-hexane extraction yielded 0.83 wt% at a temperature of 60 °C, solvent-to-solid ratio of 20 mL g⁻¹, and ultrasound intensity of 43.0 W cm^-2. The major compounds identified in the extracts were the triterpenoids lanosterol acetate and lupeol acetate and the phytosterol β-sitosterol. The supercritical CO₂ extraction yielded up to 155.74 mg β-sitosterol 100 g⁻¹ leaves at 60 °C and 200 bar. The highest antioxidant activity and total phenolic content was observed for the extract obtained by Soxhlet method, 62.87 μmol g^-1 extract and 6.05 mg g^-1 extract, respectively.

Introduction

Morus alba L. (Moraceae) is a tree species native to northern China [1] and widely distributed throughout Asia, Europe, North America, South America, and Africa [2,3]. The tree of white mulberry, as this species is also known, is of moderate size, averaging 3−6 m in height [4], and has 5−30 cm green leaves, lobed and serrated at the margins, that usually fall in winter. Its fruits, up to 5 cm in length, are green when young and purple when fully ripe [[4], [5], [6]].

The leaves of this species are nutrients for silkworms (Bombyx mori) worldwide and are used in traditional Chinese medicine to reduce blood pressure and cholesterol, treat diabetes, prevent liver damage, improve vision, strengthen joints, and facilitate urination, among other benefits [1,2,6,7]. In the phytochemical profile of the leaves of M. alba, the presence of steroids, flavonoids, amino acids, vitamins, triterpenes, tannins, anthocyanins, phenolic compounds, alkaloids, glycosides, saponins, and phytosterols is reported [2,4,6,[8], [9], [10]]. Among the phytosterols, β-sitosterol has been cited as one of the major constituents of the M. alba leaf extract obtained with apolar n-hexane and by supercritical extraction with CO₂ [11,12].

Phytosterols are lipophilic and bioactive compounds, naturally present in plant cell membranes, with regulatory function of the permeability of molecules and ions [13,14]. Their intake is critical to the significant reduction of low-density lipoprotein (LDL) cholesterol levels [15,16]. β-Sitosterol, found in the leaves of Morus alba, is one of the most abundant phytosterols, considered as a safe nutritional supplement in food with several pharmacological benefits [17], including anticancer, immunomodulatory, angiogenic, antidiabetic, antinociceptive, antimicrobial, anti-inflammatory, and antioxidant [18]. It is also employed in cosmetics such as sunscreens, moisturizers, liquid soaps, and anti-aging products [19].

Phytosterols recovery in large-scale is commonly performed from byproducts of vegetable oil (distillates) and cellulose (wood pulp/tall oil) processing [20]. The separation of the unsaponifiable matter (containing phytosterols) from these products involves distillation/evaporation at high vacuum [21]. The purification of β-sitosterol from the unsaponifiables is performed by a series of crystallization processes and by selective adsorption [22,23].

On the other hand, Soxhlet extraction is the most commonly used technique for obtaining phytosterols in laboratory scale, involving the use of organic solvents, such as n-hexane, petroleum ether, ethanol, and dichloromethane. However, studies show that alternative methods, such as extraction with supercritical CO₂ and ultrasound-assisted solvent extraction, are also effective in the recovery of these bioactive compounds [24,25].

Supercritical CO₂ extraction has the important advantage of producing solvent-free extracts after depressurization, because CO₂ is gaseous at room temperature [26]. Additionally, it is also a non-toxic, non-flammable, relatively inexpensive, and easily accessible solvent [27,28]. Regarding the second technique, the use of ultrasound in solvent extraction improves the mass transfer due to the cavitation phenomenon, providing reduced extraction time and low solvent quantity compared to the conventional Soxhlet extraction [[29], [30], [31]].

There is a lack of information regarding the extraction of M. alba leaves, especially by non-conventional methods. Thus, this study aims to evaluate the effects of temperature and pressure of supercritical CO₂ extraction on the extract yield and β-sitosterol content. In addition, the effects of temperature, volume to mass ratio, and intensity of the ultrasound-assisted n-hexane extraction on these results were also studied. The extract yield and β-sitosterol content results were compared to those of the conventional Soxhlet technique, and the antioxidant activity of the extracts of each method was determined.