Supercritical CO₂ extraction of bioactive compounds from Hibiscus sabdariffa

Highlights

• Temperature, co-solvent and pressure were evaluated by Response-surface methodology.

• The phenolic characterization of extract conditions was analyzed by HPLC-ESI-TOF-MS.

• Each chemical group showed different behavior against SFE.

• The highest phytochemical content was obtained at 50 °C, 250 bar and 16.7% ethanol.

Abstract

Hibiscus sabdariffa is used as potent phytochemical agent due to its phenolic composition. On the other hand, Supercritical Fluid Extraction (SFE) is considered a green extraction technique that provides a selective extraction using a supercritical solvent. In this work, extraction parameters such as temperature, pressure of CO₂ and percentage of co-solvent were evaluated on the chemical composition of Hibiscus sabdariffa by Response Surface Methodology (RSM). After that, individual compound concentration was determined by HPLC-ESI-TOF-MS. This design, allowed found the optimal conditions for total phenolic and organic acids content. Each chemical group showed different behavior against SFE. Despite organic acids exhibited an irregular trend; the quadratic model was fitted for total phenolic compounds. Therefore, SFE has demonstrated to be a suitable and selective technique to maximize the extraction of several phytochemical compounds from H. sabdariffa calyces.

Introduction

The increasing evidence correlating diet and chronic diseases such as diabetes, obesity, hypertension, cancer or other illnesses, made the incorporation of natural food additives one of the most important trends in the food industry. Hence, phenolic compounds are a widely studied natural family of compounds, which have demonstrated several bioactive applications, such as anti-inflammatory, antitumor, antimicrobial, anti-obesity and antioxidant functions.

In this sense, the collection of polyphenol-enriched extracts generates a great interest and attracts strong demand today. To this end, extraction process is an important step for separation and recovery of bioactive compounds from plants. In this way, supercritical fluid extraction (SFE) is considered a green extraction technique that provides attractive features overcoming many of the limitations of conventional extractions. The main advantages of SFE are the use of solvents generally recognized as safe (GRAS), lower extraction times and higher efficiency. Moreover, this technique is selective, does not require further cleaning and can be performed on small amounts of sample. SFE is the most technologically advanced extraction technique with application in environmental, chemical, food, agriculture, pharmaceutical and nutraceutical industries [[1], [2], [3]].

One of the main characteristic of SFE is the use of supercritical solvents. This type of solvents enhances the extraction rate due to their low viscosity and high diffusivity coefficient, which allow rapid penetration of the solvent into the matrix. In addition, in this technique it is possible to modify the density of the fluid by changing its pressure and / or temperature [4]. Regarding extraction fluid, carbon dioxide is widely used in SFE because of its innocuous nature to human health and environment, as well as its moderate critical temperature (31.2 °C), which permit the preservation of thermo-labile bioactive compounds. Also, this fluid prevents reactions of oxidation by avoiding the contact with air and it is readily available and reusable gas [5]. Nevertheless, the polarity of CO₂ is low and therefore, is less effective in extracting more polar phytochemical embedded in the cell wall of vegetable samples. For this reason, for the extraction of polar compounds, such as many phenolic compounds, there are polar solvent used as modifiers or co-solvents (ethanol, methanol or water) that, added in small amounts, can induce substantial changes of the solvent properties of CO₂ [2]. Among them, ethanol has been reported to be the better co-solvent due to its lower toxicity and improved extraction of phenolic compounds [6].

Besides that, operating parameters such as pressure and temperature can control the SFE process. Thus, an increase in pressure results in an increase in fluid density and enhanced solubility of the solute [5]. In addition, the combination of pressure and temperature influence on the physical properties of SFE solvent (density, viscosity and diffusivity) by mean that selectivity in extraction of target bioactive compounds can be gleaned exclusively by SFE. Generally a temperature range of 40–60 °C and a pressure range of 200–400 bar are employed in SFE for the extraction of bioactive compounds [4]. Therefore, the use of SFE represent an alternative to the extraction of natural compounds from plant origin [1,6,7].

On the other hand, Hibiscus sabdariffa (H. sabdariffa) is a tropical plant belonging to the Malvaceae family which is used as potent phytochemical agent in the therapeutic treatment of hypertension, hyperlipidemia, diabetes, obesity, inflammatory diseases among others [[8], [9], [10], [11], [12]]. These beneficial effects are based on its composition, which is rich in bioactive compounds such as hibiscus acid, phenolic acids, anthocyanins and flavonoids. Therefore, the extraction and recovery of phenolic compounds from H. sabdariffa have showed great interest from food industries to be incorporated in many functional foods or nutraceuticals.

Hence, the objective of this work was the evaluation of the extraction parameters (temperature, pressure and percentage of co-solvent) and their relationship with chemical composition of H. sabdariffa extracts. For that purpose, individual compound concentration was measured by high-performance liquid chromatography coupled to time-of-flight mass spectrometry (HPLC-ESI-TOF-MS). For SFE optimization, a response surface methodology (RSM) based on central composite design (CCD) 2³ model with 16 experiments including center and axial points was performed.