**1. Introduction**

The reddish or orange extract obtained from the seeds of annatto (*Bixa orellana* L.) has a grea<sup>t</sup> coloring strength, making this extract useful in the food and cosmetic sectors. In addition, the extract possesses inflammatory, antioxidant, and antimicrobial properties [1,2]. These pharmacological activities are due to diverse bioactive compounds such as polyphenols (i.e., hypolatin and caffeic acid) and carotenoids (i.e., bixin or 6-methyl hydrogen (9Z)-6,6-diapocarotene-6) [3]. The antioxidant activity of these compounds is attributed to their high structural conjugation and capability to couple with singlet oxygen which is responsible for denaturation of proteins belonging to cell membranes, leading to cell lysis [1,4,5].

Recent reports deal with the biological activity of annatto extract obtained by traditional techniques, which are compelling for the pharmaceutical and food companies. For instance, Zhang and collaborators assessed the ability of bixin to promote transcription of a new Ets-related factor (Nrf2) and E74-like factor 2 (ELF2) in a murine model with inflammation in the lung tissue due to SiO2 particles. This transcription factor regulates the inducible expression of numerous genes of detoxifying enzymes and antioxidants by binding to a specific DNA sequence known as the antioxidant response element (ARE), resulting in protection against several pathologies such as cancer, liver toxicity, and inflammation [6]. Further, the activation of Nrf2 in mice potentiates the antioxidant and healing abilities and decreased lung tissue inflammation after inhalation of bixin particles [2]. On the other hand, Viuda and collaborators determined the in vitro antioxidant and antimicrobial activities of annatto seed extract. They used five different hydrogen-donating or radical-scavenging tests including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable radical, ferric-reducing antioxidant power (FRAP), thiobarbituric acid reactive substance (TBARS), Rancimat, and ferrous ion (Fe2+) chelating activity. On the other hand, the microdilution in broth method was used to evaluate the antimicrobial activity against *Listeria innocua* (Spanish Type Culture Collection, University of Valencia, Research Building, Burjassot, Spain. (CECT) 910), *Aeromonas hydrophila* (CECT 5734), *Bacillus cereus* (American Type Culture Collection (ATCC) 11778), and *Pseudomona aeruginosa* (ATCC 9027). They found the annatto seed extract as an alternative preservative to replace the synthetic ones such as butyl-hydroxy toluene (BHT) in food matrices due to its extensive antioxidant activity (93.01 ± 1.22, 75.88 ± 0.21, 25.41 ± 3.52, 10.53 ± 0.31, and 1.34 ± 0.07 for DPPH (inhibition, %), TBARS (inhibition, %), FRAP (TEAC), FIC (chelating effect, %), and rancimat (AAI), respectively. Conversely, it had a mild antimicrobial action compared to some synthetic antimicrobials (1.024 mg/mL, 256 mg/mL, 512 mg/mL, and 256 mg/mL for *P. aeruginosa, B. cereus, L. innocua,* and *A. hydrophila*, respectively) [1].

Maceration, leaching, extraction with supercritical fluids (CO2), dispersive liquid–liquid, sonication, enzymatic extraction, microextraction, and microwave-assisted extraction (MAE) are the most widely used methods for the extraction of bioactive molecules from plants [7]. However, the extraction technique affects the functional activities of the extract and thus, conventional approaches require long processing times, exposing the active compounds to degradation and resulting in low efficiency. On the other hand, some emerging extraction techniques such as supercritical CO2 extraction are costly and involve high energy consumption [8]. Further, the use of mechanical methods, spouted bed, sonication, microwaves, and supercritical fluids (CO2) as emerging extraction technologies, have been focused simply on the extraction of bixin and norbixin [7,9–11]. Most of these studies highlight the coloring capacity of the extract, and others evaluate the effect of the extraction technique on the antioxidant and antimicrobial activities separately [11,12].

The microwave-assisted extraction (MAE) involves the formation of high-energy electromagnetic waves with a frequency, oscillation period, and wavelength ranging from 300 MHz–30 GHz, 3 × 10−<sup>9</sup> s–33 × 10−<sup>12</sup> s, and 1–10 mm, respectively. These waves have the ability to change the molecular rotation and ionic mobility of the medium without altering the sample. This rotation generates the adsorption and dissipation of energy in the medium, causing a generalized heating, forcing the rapid migration of all the active compounds from the solid-phase to the solvent-phase [7,13–17]

For this reason, the goal of this study was to define the optimal microwave-assisted extraction (MAE) conditions to render the bioactive compounds of annatto seeds without affecting their antioxidant and antimicrobial activities. The solvent concentration, pH, treatment time and solvent-to-seed ratio were taken as independent variables employing a surface response experimental design.

#### **2. Materials and Methods**
