We designed both single and multiple factors experiments. The Response Surface Methodology (RSM) with five-level three-factor central composite rotatable design (CCRD) was applied to determine the optimal combination of solid-to-liquid ratio, extraction time, and temperature that yield the maximum amount of total polyphenols. For this experiment, distilled water was used as an extraction solvent.

In a series of single-factor experiments (SFEs), each factor was assessed to give an experimental range. The effect of temperature on polyphenols’ extraction was determined by conducting triplicates SFE in a temperature range of 27, 40, 50, 60, 70, 80, and 90°C while other factors -time and solid-to-solvent ratio were fixed at 1 hour and 1:20 respectively. The temperature that gave the maximum yield of total polyphenols was used in the subsequent experiment. In the next experiment, the time in hours was varied; (0.5, 1, 2, 3, 5) while the solid-to-solvent ratio was maintained at 1:20. Finally, both the optimal time and temperature from previous experiments were used to establish the best solid-to-solvent ratio from the range of 2.5%, 3.3%, 5%, and 10%. Later, the RSM designed a multiple factor experiment (MFE) that had 20 experiments. The factors and levels were determined from the SFEs (Table 1).

The 20 experiments were grouped into two blocks with total polyphenols content (TPC) as a response variable. The first block includes eight factorial points and four center points. The second block utilizes the six axial points of the design and two replicas at the center. The six replicas at the center were added to estimate the pure error sum of squares. The distance from the center (α) determines the extreme points, i.e., the lowest and highest values of the factors. With CCDR, the distance is 1.68179. The experiments were randomized to minimize unexplained variability in the observed response due to extraneous factors. Finally, confirmation experiments for the optimized extraction conditions for aqueous extraction of total polyphenols from fruits of A. esculentus were performed. The extract so obtained was subsequently standardized.

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